Are we not liposomes? Hot-Zone and the De-evolution of the liposome concept
Scott C. Hartsel
Department of Chemistry, University of Wisconsin - Eau Claire, Eau Claire, Wisconsin, U.S.A.20
CD Spectra with Serum Components
DISCUSSION
600
OBJECTIVE
The objective of this study was to explore the
stability, activity, binding and interactions of the
heat-induced superaggregated form of
Amphotericin B (HFZ)as compared to conventional
Fungizone. In addition, our third proposition, that
AmB may be toxic in part due to its stimulation of
cytokines, was explored in a human monocyte cell
culture.
•Fungizone and Hot-Zone were scanned using an Applied Photophysics
SK 17 stopped flow spectrometer with CD attachment and diode array
detector.
For 30 years the “liposome concept” (microencapsulations of
drugs in plain or modified liposomes) has held great promise as a
way to selectively target and reduce the toxicity of therapeutic
agents. Amphotericin B (AmB) can be viewed as the first major
liposome success story. No fewer than three new liposomal or
lipid associated forms of Amphotericin are on the market and all of
them present a significant improvement of therapeutic index over
the very toxic micellar form, Fungizone. Interestingly, however,
none of these forms really correspond to the original concept of
liposome as an encapsulation of a drug, but rather they are novel
supramolecular aggregates of AmB and lipid(s) that change the
distribution, activity and thermodynamic stability of the drug
complexes. Hence, it may be possible to more easily reduce the
toxicity of AmB by complexing it with a single agent in stable
nonliposomal, non-membrane complexes. In fact, Amphocil, the
cholesteryl sulfate:AmB formulation of AmB, does just this.
FZ10uMPBS
HFz10uMPBS
-400
HFZ10uMHSA
-600
300
RESULTS
320
500
340
360
380
400
Wavelength
420
10uM FZ and HFZ in PBS or Human
Serum. Human Serum filtered and diluted
1:2 in PBS. Samples incubated at 37C for
15min before scans.
400
300
MATERIALS AND METHODS
440
460
Whole Serum
KInetics (1:5 serum)
200
100
0
Whole Serum
FZ-HS
HFZ-HS
HFZ in PBS
-300
-400
300
•Serum distribution studies show that Hot-Zone dissociates and
distributes less completely and more slowly into lipoprotein fractions,
serum and albumin alone. This alteration in distribution may effect toxicity
by lessening uptake into tissues via LDL receptors and by maintaining an
aggregate state (HFZ) which may be taken up by the reticuloendothelial
system as proposed for commercial liposomal systems.
FZ10uMHSA
-200
FZ in PBS
320
340
360
380
400
Wavelength
420
440
460
Figure 2 A and B. CD Spectra of HFZ and FZ in the presence of
Human serum and albumin. The CD doublet is indicative of
self-associated AmB. Note the great stability of HFZ in the
presence of serum and albumin (indicated by doublet stability)
•Circular dichroism studies show that a larger fraction of AmB from
Fungizone dissociates in the presence of serum and serum albumin
whereas proportionally less Hot-Zone redistributes. Hot-Zone thus has a
more stable aggregate “core” supramolecular structure and interacts less
with serum lipoproteins than the AmB/deoxycholate micelles of
Fungizone.
•Despite a generally greater amount of AmB aggregate in Hot-Zone as
measured by absorption spectroscopy (PharmSci, Volume 1, Issue
4,October - December 1999 ), its activity against cholesterol-containing
model membranes is markedly lower than Fungizone while activity against
model fungal membranes is essentially the same (see model below). This
suggests a more “inert” aggregate is promoted by heating and it may be
the source of the reduced toxicity of Hot-Zone in animal model studies. In
addition, its toxicity may be reduced by provoking a smaller cytokine
response. Its efficacy in animal disease models indicates that AmB
monomers can still reach their targets, i.e fungal or Leishmania cells.
collaboration
with K. Wasan)
INTRODUCTION
-200
-100
Persistence of the Hot-Zone Structure
Lipoprotein Distribution Studies (in
0
TNF AssayA THP-1 (ATCC TIB 202) human monocyte cells were grown in RPMI
1640 medium containing 10% low pyrogen fetal calf serum, 100 u
penicillin, 100 µg/mL streptomycin and 1 µg/mL gentamycin. The cells
were a high TNF producing strain cultured continuously in 1988. The
cells were cultured in suspension at 37˙C and 5% CO2. The cells
required 6-8 days to reach their terminal density. Cells were centrifuged
and placed in fresh media at the outset of the experimental exposure to
FZ and HFZ. The ELISA assay for TNF-alpha was a sandwich-type
assay with alkaline phosphatase/pNPP colorimetric detection from a kit
from Immunotech (purchased from Sigma Chemical, St.Louis, MO).
HSA
200
²e(r-l)
Fungizone, a formulation of Amphotericin B and deoxycholate, is a toxic, yet
effective, antifungal agent used in treating systemic fungal infections. When
treated by mild heating (70° C for 20 minutes) Fungizone develops a blue
shifted spectrum accompanied by an increase in the size of the complexes
("Hot-Zone"). This form also can be induced in Amphotericin solutions
without deoxycholate. The distinct absorption and CD spectra suggest that
Hot-Zone has a new and different supramolecular structure. Absorption
spectra and HPLC demonstrate that there is little or no chemical degradation
from this process. Lyophilization and reconstitution of Hot-Zone shows that
the unique CD spectrum of this form is persistent. Others have demonstrated
that Hot-Zone reduces side effects and toxicity in animal disease models in a
manner reminiscent of expensive and complex liposomal Amphotericin
formulations. We have attempted to pinpoint the mechanisms of this reduced
toxicity. Kinetic and CD studies show a major increase in the stability of the
Hot-Zone complex in the presence of serum albumin and lipoproteins as
compared to Fungizone. Preliminary studies using THP-1 human monocytes
show that Hot-Zone provokes a smaller release of TNF, which is associated
with the unpleasant side effects of Amphotericin. In addition, the Hot-Zone
complex itself is intrinsically less membrane active against model
mammalian membranes, but equally effective against model fungal
membranes. Thus Hot-Zone may be a simple, less toxic and very cheap
alternative to Fungizone and costly new liposomal preparations. The
simplicity of the system is also helpful in determining what features are most
important in improving the therapeutic index of Amphotericin and guide the
design of new formulations. Due to the ease of preparation, Hot-Zone may
be especially attractive for treatment of AIDS patients in third world countries
where liposomal preparations may be unavailable.
What are the key factors which determine the toxicity of
AmB preparations? We propose that there are essentially
three intertwined factors which influence the toxicity and
efficacy of AmB preparations: 1) direct membrane activity
via ion channel formation, 2) differences in distribution
and delivery to tissues due to differences in serum
lipoprotein or protein binding, and 3) initiation of an
inflammatory cytokine response.
10 uM FZ and HFZ in PBS or Human Serum Albumin
(Sigma Chemical). HSA diluted with PBS 15mg/mL.
Samples incubated at 37 C for 15 min before scans.
400
CD (²e(l-r))
ABSTRACT
Circular Dichroism and Absorbance Studies
Membrane Ion Channel Forming Activity
•Fungizone or Hot-Zone at 20 g per ml of human plasma were incubated
at 37ºC for 60 minutes
Model for “Hot-Zone” Membrane-Activity
Macrophage reservoir?
CHOLESTEROL/egg
PC MEMBRANES
•The plasma samples were separated into their high-density lipoprotein
(HDL), low-density lipoprotein (LDL), triglyceride-rich lipoprotein (TRL), and
lipoprotein deficient (LPD) fractions by density gradient ultracentrifugation
•Each lipoprotein and LPDP sample was assayed for Amphotericin B using
High Performance Liquid Chromatography (HPLC)
Fungizone
active
AmB
oligomer
"Hot-Zone"
AmB
monomer
Statistical analysis
Data was analyzed by one way analysis of variance (GraphPad Instat).
Critical differences were assessed by Tukey-Kramer post hoc test.
Differences were considered significant if p < 0.05. All data are expressed
as means ± standard deviation.
Ion Currents
Figure 1. Hot-zone (HFZ) has persistence; CD spectra of
reconstituted lyophilized preps (after 2 weeks at -20 C)are
similar to the distinct CD of fresh HFZ preps with very little
reversion. Long term experiments are underway.
Lyophilization in 5% dextrose instead of PBS shows even less
change upon reconstitution(not shown).
ERGOSTEROL/egg
PC MEMBRANES
AmB channel w/
cholesterol or no sterol
Binding to Serum Components
Figure 3 A and B. Fluorescence detected ion currents from
model mammalian and fungal vesicles. HFZ is about equally
effective against model fungal vesicles but is much less
potent vs. model mammalian vesicles.
As another potentially even simpler and less inexpensive
alternative to lipid-associated forms of AmB, Bolard and Gaboriau
(CNRS) have shown that AmB or Fungizone solutions may be
treated with moderate heat (70oC for 20 minutes) to produce a
new self-associated state of AmB, the “superaggregate”, which we
will refer to as heat-treated Fungizone (HFZ) or "Hot-Zone". This
new species is spectroscopically distinct from fresh AmBdeoxycholate suspensions (Fungizone, FZ), having a blue-shifted
absorption maximum and characteristic CD spectrum. In model
murine infections, Bolard, Gaboriau and co-workers have shown
that HFZ has a superior therapeutic index to FZ. This
superaggregate species is fascinating both for the possible
practical therapeutic benefits and for its potential for explaining
the root mechanisms for toxicity reduction in the simplest possible
improved Amphotericin B drug delivery system.
Cytokine Induction
Pyranine fluorescence method for detecting electrogenic ion
currents caused by channel forming compounds.
Table 1. Static Distribution of Fungizone and Hot-Zone at 20 g
of AmB per ml within human plasma following incubation for 60
minutes at 37C. Data expressed as mean  standard deviation
(n=3). Most HFZ remains in LPDP, but it is largely selfassociated as compared to FZ aggreagtes which are
“dismantled” (see below).
AmB/sterol channel w/
ergosterol
CONCLUSION
Heat-induced superaggregation of Amphotericin B may represent an
simple, viable alternative to the toxic parent product, Fungizone, for
the same reasons as liposomal preparations. Thus it represents a
simplification or “de-evolution” of the liposome concept in this case to
a prep which reduces the chemical potential and distribution of AmB by
changing its molecular aggregation. It remains to be seen whether such
a preparation would be safe and effective and have a better therapeutic
index in human disease. Areas of concern would be the size of the
aggregates and their stability.
ACKNOWLEDGEMENTS
Figure 4. Induction of TNF-a by FZ and HFZ. At 5µM FZ
induces nearly 3X as much TNF as HFZ.
This project was carried out with UW-EC undergrad students Brad Baas,
Angela Scott , L.Tuck Foree, Hilary Preis and Emily Bauer with Evan
Kwong , Manisha Ramaswamy and Kishor M. Wasan from the University of
British Columbia. It was supported with funding from the Medical Research
Council of Canada (grant # MT-14484 to KMW) and the NSF (MCB9603582 to SCH).