Presentation on theme: "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,"— Presentation transcript:
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.20ABSTRACT 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. INTRODUCTION 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. 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 (70 o C 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 AmB- deoxycholate 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. MATERIALS AND METHODS Lipoprotein Distribution Studies (in collaboration with K. Wasan) Fungizone or Hot-Zone at 20 g per ml of human plasma were incubated at 37ºC for 60 minutes 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) 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 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 self- associated as compared to FZ aggreagtes which are “dismantled” (see below). DISCUSSION 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. 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. ACKNOWLEDGEMENTS 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 (MCB- 9603582 to SCH). RESULTS 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. Pyranine fluorescence method for detecting electrogenic ion currents caused by channel forming compounds. Circular Dichroism and Absorbance Studies Fungizone and Hot-Zone were scanned using an Applied Photophysics SK 17 stopped flow spectrometer with CD attachment and diode array detector. TNF Assay- A 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% CO 2. 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). CHOLESTEROL/egg PC MEMBRANES ERGOSTEROL/egg PC MEMBRANES Binding to Serum Components Membrane Ion Channel Forming Activity Whole Serum Model for “Hot-Zone” Membrane-Activity 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. 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). Persistence of the Hot-Zone Structure CD Spectra with Serum Components Cytokine Induction HSA Whole Serum 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) 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. Figure 4. Induction of TNF- by FZ and HFZ. At 5µM FZ induces nearly 3X as much TNF as HFZ. Macrophage reservoir? 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.