Kinetic fluorescent methods for measuring functional delivery of

Kinetic fluorescent methods for measuring functional delivery of membrane active antibiotics or Dr. Scott C. Hartsel University of Wisconsin-Eau Claire

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

What is Amphotericin B? Binds weakly to Am. B Nystatin Cholesterol: humans Binds strongly to Am. B Amphotericin B Ergosterol: fungi

Q: How can you reduce toxicity? A: Associate with liposomes. Bolard’s model • Reducing effective chemical potential of Am. B 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 (70 o. C, 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

A Simple Solution: Hot Zone • We wanted to find out “How and Why? ” by asking: –How has the structural arrangement of Am. B 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?

Hot-Zone-Analysis Applied Photophysics Stopped-Flow diode array and conventional spectrophotometer, spectrofluorimeter, and circular dichroism (CD) spectrometer

Hot-Zone-Absorption Spectra HEAT

CD Spectra (circular dichroism)

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. Absorption Out of phase In phase

CD Spectra • Am. B molecules normally have no CD spectrum in the visible light region, but when selfassociated (oligomers)they have intense CD spectra. The dimer is the minimal unit of CD activity. • This property gives a very sensitive handle on Am. B’s supramolecular geometry and changes in that structure. - +

Hot-Zone-CD Spectra • Circular dichroism: sensitive to small changes in supramolecular structure HEAT -

Hot-Zone-CD Spectra • Circular dichroism: sensitive to small changes in supramolecular structure -

Persistence of Hot-Zone. Lyophilization studies show stability

Experimental System Membrane Activity of Hot-Zone Extruded 1000Å Liposome Membrane Vesicles • • Membranes with 10% ergosterol are fungal models; with cholesterol they are mammalian models. With KCl gradient K+ permeation creates a voltage, (K+ selective for Am. B). H+ equilibrates with Pyranine fluorescence responds to p. H linearly from ~6. 2 -7. 8. Fluorescence decrease means net cation (K+) selectivity. Amphotericin High K+ , Cl 2 m. M pyranine H+ K+ - + + + Low K+, Cliso-osmotic H+ Fluorescence Decrease

Hot-Zone/Membrane Channel Activity Model mammalian membranes with cholesterol Hot-Zone has much less activity Model fungal membranes With ergosterol Hot-Zone has similar activity !

Membrane Activity in the Presence of Serum Components Change in p. H versus Time showing fluorescence detected ion currents on model mammalian membranes comparing Fungizone and Hot-Zone in the presence of 15 mg/m. L human serum albumin in external buffer (315 m. M sucrose, 15 m. M K 2 HPO 4, p. H 7. 20 at 37 C)

Hot-Zone-Kinetic Stability (HSA)* 500 Fungizone aggregates are destabilized by serum albumin -500 sec/37 C 0 Hot-zone aggregates are much more stable in the presence of serum albumin

Hot-Zone-Kinetic Stability (HSA)* • Extra stability of Hot-Zone probably buys enough time so that Am. B 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

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?