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?