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

4. 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

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

6. 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

7. 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

8. 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

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

10. Hot-Zone-Absorption Spectra HEAT

11. CD Spectra (circular dichroism)

12. 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 phase Out of phase

13. 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. - +

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

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

16. Persistence of Hot-Zone- Lyophilization studies show stability

17. 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 System Extruded 1000Å Liposome Membrane Vesicles High K+, Cl- Low K+, Cl- iso-osmotic H+ Amphotericin 2 mM pyranine K+ H+ Membrane Activity of Hot-Zone ++++++ ------ Fluorescence Decrease

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

19. 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 K 2 HPO 4, pH 7.20 at 37  C) Membrane Activity in the Presence of Serum Components

20. 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

21. 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

22. Engulfing and slow release from macrophages

23. 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

24. A Happy Ending?