Antileishmanial activity, cytotoxicity and cellular response of amphotericin B in combination with crotamine derived from Crotalus durissus terrificus venom using in vitro and in silico approaches.

OBJECTIVE: To investigate the in vitro activity, synergism, cytotoxicity and cellular immunological response, as well as the molecular affinity between amphotericin B (AmB) and crotamine (CTA), derived from Crotalus durissus terrificus venom against Leishmania amazonensis. METHODS: This study performed the inhibition of promastigotes and amastigotes' growth under different concentrations of the drug and pharmacological combinations (AmB + CTA) based on the Berimbaum method (synergism study). The lactate dehydrogenase (LDH) quantification method was used to determine the cytotoxicity of the drug and combinations employing four cell lines (J774, HepG2, VERO, and C2C12). Following, the levels of Tumour Necrose Factor-alpha (TNF-α) and Interleukin-12 (IL-12) cytokines, using enzyme-linked immunosorbent assay (ELISA) and nitrites, as an indirect measure of Nitric Oxide (NO), using the Griess reaction were assessed in the supernatants of infected macrophages. In silico approach (molecular docking and dynamics) and binding affinity (surface plasmon resonance) between the drug and toxin were also investigated. RESULTS: CTA enhanced AmB effect against promastigote and amastigote forms of L. amazonensis, decreased the drug toxicity in different cell lines and induced the production of important Th1-like cytokines and NO by infected macrophages. The pharmacological combination also displayed consistent molecular interactions with low energy of coupling and a concentration-dependent profile. CONCLUSION: Our data suggest that this pharmacological approach is a promising alternative treatment against L. amazonensis infection due to the improved activity (synergistic effect) achieved against the parasites' forms and to the decreased cytotoxic effect.

Antifungal Liposomes Directed by Dectin-2 Offer a Promising Therapeutic Option for Pulmonary Aspergillosis.

Invasive fungal diseases cause millions of deaths each year. There are currently approximately 300,000 acute cases of aspergillosis, most of which result from a pulmonary infection of immunocompromised patients by the common soil organism and opportunistic pathogen Aspergillus fumigatus Patients are treated with antifungal drugs, such as amphotericin B (AmB). However, AmB has serious limitations due to human organ toxicity. AmB is slightly less toxic if loaded in liposomes, such as AmBisome or AmB-loaded liposomes (AmB-LLs). Even with antifungal therapy, recurrent infections are common, and 1-year fatality rates may exceed 50%. We have previously shown that coating AmB-LLs with the extracellular oligomannan-binding domain of the C-type lectin receptor Dectin-2 (DEC2-AmB-LLs) effectively targets DEC2-AmB-LLs to cell walls, exopolysaccharide matrices, and biofilms of fungal pathogens in vitroIn vitro, DEC2-AmB-LLs reduce the effective dose of AmB for 95% inhibition and killing of A. fumigatus 10-fold compared to that of untargeted AmB-LLs. Herein we tested the antifungal activity of DEC2-AmB-LLs relative to that of untargeted AmB-LLs in immunosuppressed mice with pulmonary aspergillosis. Remarkably, DEC2-AmB-LLs bound 30-fold more efficiently to A. fumigatus at sites of infection in the lungs. Furthermore, Dectin-2-targeted liposomes delivering AmB at a dose of 0.2 mg/kg of body weight significantly reduced the fungal burden in lungs compared to results with untargeted AmB-LLs at 0.2 mg/kg and micellar voriconazole at 20 mg/kg and prolonged mouse survival. By dramatically increasing the efficacy of antifungal drugs at low doses, targeted liposomes have the potential to create a new clinical paradigm to treat diverse fungal diseases.IMPORTANCE Invasive aspergillosis (IA) generally results from a pulmonary infection of immunocompromised patients by the common soil organism and opportunistic pathogen Aspergillus fumigatus The susceptible population has expanded rapidly due to the increased number of cancer patients with immunocompromising chemotherapy and transplant patients taking immunosuppressants. Patients are treated with antifungals, such as liposomal amphotericin B, with per-patient costs exceeding $50,000 in the United States. However, AmB has serious side effects due to host toxicity, which limits its usage and contributes to the lack of fungal clearance in patients at safe doses. Fifty percent of IA patients die within a year. Herein, we employed liposomal amphotericin B coated with the innate immune receptor Dectin-2 to direct antifungals specifically to the fungal pathogen. Using two mouse models of pulmonary aspergillosis, we demonstrate that Dectin-2-targeted delivery of amphotericin B to A. fumigatus resulted in remarkably higher efficacy than that of the untargeted antifungal formulations.

Biopharmaceutical Assessment and Irritation Potential of Microemulsions and Conventional Systems Containing Oil from Syagrus cearensis for Topical Delivery of Amphotericin B Using Alternative Methods.

The aim of this study was to compare the biopharmaceutical characteristics and irritation potentials of microemulsions (MEs) and conventional systems (CSs) containing oil from Syagrus cearensis for topical delivery of Amphotericin B (AmB). Pseudo-ternary phase diagrams were constructed using a water titration method to develop the MEs, and the CSs were prepared according to the classical technique of phase inversion. In the skin permeation and retention study, dermatomed pig skin without stratum corneum was used as an alternative disturbed skin model. The irritation potential was evaluated using three different methods, chorioallantoic membrane assays (HET-CAM and CAM-TBS), and bovine corneal opacity and permeability (BCOP) test. The optimized formulation (ME1) consisting of 0.1% (w/w) Amphotericin B, 9.1% (w/w) catolé oil, 81% (w/w) Smix (1:1, Tween 20 and Kolliphor EL) possessed droplet size of 31.02 ± 0.9 nm, zeta potential of -23.4 mV, and viscosity 0.63 ± 0.1 Pa.s. ME1 exhibited greater retention of AmB in to skin layers (84.79 ± 2.08 µg cm-2) than all the others formulations. In general, MEs showed higher drug release and retention than CSs and all of the formulations showed greater retentivity than permeability. Only MEs developed using Labrasol/Plurol Oleique (L/PO) as the surfactant and co-surfactant exhibited a moderate irritation potential; all other MEs and CSs were classified as non-irritants or slight irritants. The results indicate that formulations containing oil from S. cearensis are promising alternatives for the delivery of AmB targeting the treatment of cutaneous leishmaniasis.

Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosaisolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosaisolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.

Surface-engineered dendrimeric nanoconjugates for macrophage-targeted delivery of amphotericin B: formulation development and in vitro and in vivo evaluation.

The present study aimed to develop an optimized dendrimeric delivery system for amphotericin B (AmB). Fifth-generation (5.0 G) poly(propylene imine) (PPI) dendrimers were synthesized, conjugated with mannose, and characterized by use of various analytical techniques, including Fourier transform infrared spectroscopy (FTIR), (1)H nuclear magnetic resonance ((1)H-NMR) spectroscopic analysis, and atomic force microscopy (AFM). Mannose-conjugated 5.0 G PPI (MPPI) dendrimers were loaded with AmB and evaluated for drug loading efficiency, in vitro drug release profile, stability, hemolytic toxicity to human erythrocytes, cytotoxicity to and cell uptake by J774A.1 macrophage cells, antiparasitic activity against intracellular Leishmania donovani amastigotes, in vivo pharmacokinetic and biodistribution profiles, drug localization index, toxicity, and antileishmanial activity. AFM showed the nanometric size of the MPPI dendrimers, with a nearly globular architecture. The conjugate showed a good entrapment efficiency for AmB, along with pH-sensitive drug release. Highly significant reductions in toxicity toward human erythrocytes and macrophage cells, without compromising the antiparasitic activity of AmB, were observed. The dendrimeric formulation of AmB showed a significant enhancement of the parasiticidal activity of AmB toward intramacrophagic L. donovani amastigotes. In the in vitro cell uptake studies, the formulation showed selectivity toward macrophages, with significant intracellular uptake. Further pharmacokinetic and organ distribution studies elucidated the controlled delivery behavior of the formulation. The drug localization index was found to increase significantly in macrophage-rich organs. In vivo studies showed a biocompatible behavior of MPPIA, with negligible toxicity even at higher doses, and promising antileishmanial activity. From the results, we concluded that surface-engineered dendrimers may serve as optimized delivery vehicles for AmB with enhanced activity and low or negligible toxicity.

Aerosolized liposomal amphotericin B: prediction of lung deposition, in vitro uptake and cytotoxicity.

To predict the efficacy and toxicity of pulmonary administration of liposomal amphotericin B (L-AMB) for the treatment or the prevention of pulmonary invasive aspergillosis, a multistage liquid impinger was used to estimate the concentrations of drug that could be attained in different lung compartments after nebulization. The highest concentration of amphotericin B was found in the alveolar compartment, where it was calculated that the concentration in the lung surfactant could reach 54 µM or more when 21.6 µmoles of drug as liposomes was nebulized. The uptake and toxicity of L-AMB were studied in vitro using the A549 human lung epithelial cell line. Uptake was time and concentration-dependent and reached intracellular concentrations exceeding the minimal inhibitory concentrations for most Aspergillus species. The toxicity of L-AMB toward these cells, estimated by the MTT reduction assay, was reduced compared with the conventional form, deoxycholate amphotericin B (D-AMB), with an IC(50) value of about 120 µM after 24 h of exposure for D-AMB, but only a 13% reduction in viability for 200 µM L-AMB at 24 h. These results indicate that aerosol therapy with nebulized L-AMB could be efficient but that doses need to be carefully controlled to avoid toxicity.

Antimycotic-antibiotic amphotericin B promotes influenza virus replication in cell culture.

In general, antibiotics are not rated as substances that inhibit or support influenza virus replication. We describe here the enhancing effect of the polyene antibiotic amphotericin B (AmB) on influenza virus growth in Vero cells. We show that isolation rates of influenza A and B viruses from clinical samples can be dramatically enhanced by adding AmB to the culture medium. We demonstrate that AmB promotes the viral uptake and endocytic processing of the virus particles. This effect is specific for Vero and human nasal epithelial cells and was not observed in Madin-Darby canine kidney cells. The effect of AmB was subtype specific and more prominent for human seasonal influenza strains but absent for H5N1 human viruses. The AmB-enhancing effect seemed to be solely due to the viral hemagglutinin function. Our results indicate that the use of AmB may facilitate influenza virus isolation and production in Vero cells.

Effects of caspofungin, Candida albicans and Aspergillus fumigatus on toll-like receptor 9 of GM-CSF-stimulated PMNs.

The possible involvement of Toll-like receptors (TLRs) 1, 2, 4 and 9 in the interaction of antifungal drugs with polymorphonuclear neutrophils (PMNs) in response to Aspergillus fumigatus and Candida albicans as stimuli was investigated. Caspofungin revealed the broadest capacity to enable C. albicans and A. fumigatus to stimulate TLR upregulation, TLR 2 by A. fumigatus and TLRs 4, 9 by C. albicans. Conventional amphotericin B (cAMB) stimulated only A. fumigatus to induce TLRs 2 and 4 upregulation; voriconazole stimulated A. fumigatus and fluconazole C. albicans to induce TLR 9 upregulation. For cAMB, only TLR 9 was upregulated by A. fumigatus, whereas in the case of voriconazole, TLRs 2, 4, 9 were upregulated. Caspofungin revealed the broadest capacity: C. albicans was stimulated to upregulate TLRs at least at one of the concentrations, and A. fumigatus was stimulated to upregulate TLRs 2, 4. TLR 9 was upregulated two to three fold by all antifungal drugs on protein, except for fluconazole at the RNA level. Candida albicans preincubated with caspofungin has additional effects on CD11b expression and IL8 chemotaxis in CpG-DNA-stimulated PMNs. These results indicate a relevant upregulation with a functional relevance of TLR 9 in the presence of C. albicans strains preincubated with caspofungin at three concentrations.

A putative P-type ATPase, Apt1, is involved in stress tolerance and virulence in Cryptococcus neoformans.

The export of virulence factors, such as the capsule polysaccharide, to the cell surface is a critical aspect of the pathogenicity of Cryptococcus neoformans. A view of capsule export via exocytosis and extracellular vesicles is emerging, but the molecular mechanisms underlying virulence factor transport pathways remain to be established. In this study, we characterized the APT1 gene, which encodes a predicted integral membrane P-type ATPase belonging to the type IV, Drs2 family of aminophospholipid translocases (flippases) (APTs). APTs maintain the phospholipid asymmetry that is critical in membrane fusion events for trafficking and in establishing cell polarity. Deletion of the APT1 gene resulted in phenotypes consistent with similar roles in C. neoformans. These included altered actin distribution, increased sensitivity to stress conditions (oxidative and nitrosative stress) and to trafficking inhibitors, such as brefeldin A and monensin, a reduction in exported acid phosphatase activity, and hypersensitivity to the antifungal drugs amphotericin B, fluconazole, and cinnamycin. However, there was no difference in growth, capsule size, or melanin production between the wild type and the apt1 mutant strains at either 30 degrees C or 37 degrees C. Despite the absence of an influence on these major virulence factors, Apt1 was required for survival during interactions with macrophages, and apt1 mutants exhibited attenuated virulence in a mouse inhalation model of cryptococcosis. Therefore, Apt1 contributes to virulence and the stress response in C. neoformans through apparent functions in membrane fusion and trafficking that do not influence the deposition of major virulence factors, such as capsule and melanin, outside the cell.

Effects of immunomodulatory and organism-associated molecules on the permeability of an in vitro blood-brain barrier model to amphotericin B and fluconazole.

Amphotericin B (AMB) is used to treat fungal infections of the central nervous system (CNS). However, AMB shows poor penetration into the CNS and little is known about the factors affecting its permeation through the blood-brain barrier (BBB). Therefore, we studied immunomodulatory and organism-associated molecules affecting the permeability of an in vitro BBB model to AMB. We examined the effects of interleukin-1 beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), lipopolysaccharide (LPS), lipoteichoic acid (LTA), zymosan (ZYM), dexamethasone (DEX), cyclosporine, and tacrolimus on transendothelial electrical resistance (TEER); endothelial tight junctions; filamentous actin; and permeability to deoxycholate AMB (DAMB), liposomal AMB (LAMB), and fluconazole. Proinflammatory cytokines and organism-associated molecules significantly decreased the mean TEER by 40.7 to 100% (P < or = 0.004). DEX increased the mean TEER by 18.2 to 26.4% (P < or = 0.04). TNF-alpha and LPS increased the permeability to AMB by 8.2 to 14.5% compared to that for the controls (1.1 to 2.4%) (P < or = 0.04). None of the other molecules affected the model's permeability to AMB. By comparison, the BBB model's permeability to fluconazole was >78% under all conditions studied, without significant differences between the controls and the experimental groups. LPS and TNF-alpha decreased tight-junction protein zona occludens 1 (ZO-1) between endothelial cells. In conclusion, IL-1beta, ZYM, and LTA increased the permeability of the BBB to small ions but not to AMB, whereas TNF-alpha and LPS, which disrupted the endothelial layer integrity, increased the permeability to AMB.

Amphotericin B.

Invasive fungal infections are a major cause of morbidity and mortality in immunodeficient individuals (such as AIDS patients) and in transplant recipients or tumor patients undergoing immunosuppressive chemotherapy. Amphotericin B is one of the oldest, yet most efficient antimycotic agents. However, its usefulness is limited due to dose-dependent side-effects, notably nephrotoxicity. In order to improve its safety margin, new pharmaceutical formulations of amphotericin B have been designed especially to reduce its detrimental effects on the kidneys. Since the 1980s, a wide variety of new amphotericin B formulations have been brought forward for clinical testing, many of which were approved and reached market value in the 1990s. This review describes and discusses the molecular genetics, pharmacological, toxicological, and clinical aspects of amphotericin B itself and many of its innovative formulations.

Interaction of amphotericin B and its low toxic derivative, N-methyl-N-D-fructosyl amphotericin B methyl ester, with fungal, mammalian and bacterial cells measured by the energy transfer method.

Amphotericin B (AMB) derivative, N-methyl-N-D-fructosyl amphotericin B methyl ester (MFAME) retains the broad antifungal spectrum and potency of the parent antibiotic, whereas its toxicity towards mammalian cells is reduced by about two orders of magnitude. The purpose of this work was to find out whether the differences observed in the toxicity of MFAME and native AMB are due to the differential drugs affinity to fungal and mammalian cell membranes. Comparative studies on AMB and MFAME biological activity and their affinity to fungal, mammalian and bacterial cells were performed. The interaction of AMB and MFAME with cells have been studied by fluorescence method based on the energy transfer between membrane fluorescent probe (donor) and the polyenic chromophore of the antibiotic (acceptor) simultaneously present in the cell membrane. The amount of the antibiotic bound to cells was indicated by the extent of fluorescence quenching of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) or 1,6-diphenyl-1,3,5-hexatriene (DPH) by polyenic chromophore of the antibiotic. The results obtained indicate that binding extent and characteristics for both antibiotics are comparable in the three types of cells studied. Dramatically lower toxicity of MFAME as compared to AMB towards mammalian cells is not related to the antibiotic-cell affinity, but rather to different consequences of these interactions for cells, reflected in membrane permeabilization. MFAME is definitely less effective than parent AMB in the permeabilizing species formation in mammalian cell membrane.

Utility of poly(ethylene glycol) conjugation to create prodrugs of amphotericin B.

This paper reports on the synthesis, safety, and efficacy of a series of water-soluble derivatives of poly(ethylene glycol) (PEG)-conjugated amphotericin B (AmB). PEG 40 000 attached to the sugar amino group of AmB via labile carbamate and carbonate linkages was examined. The synthetic program conducted for this investigation provided a series of disubstituted PEG-AmB derivatives which had in vitro PEG half-life of hydrolyses rates in rat plasma varying between 1 and 3 h. Importantly, all conjugates demonstrated less than 6% hydrolysis following 24 h incubation in pH 7.4 phosphate buffer at 25 degrees C and showed solubilities greater than 46 mg/mL in aqueous solutions. The solubility of AmB in the conjugates increased up to approximately 200 times compared to unmodified AmB in saline. As a major finding, this investigation demonstrated that conjugation of PEG to AmB could produce conjugates that were significantly (6x) less toxic than AmB-deoxycholate and maintained, or even had enhanced, in vivo antifungal activity.

Drs2p-related P-type ATPases Dnf1p and Dnf2p are required for phospholipid translocation across the yeast plasma membrane and serve a role in endocytosis.

Plasma membranes in eukaryotic cells display asymmetric lipid distributions with aminophospholipids concentrated in the inner and sphingolipids in the outer leaflet. This asymmetry is maintained by ATP-driven lipid transporters whose identities are unknown. The yeast plasma membrane contains two P-type ATPases, Dnf1p and Dnf2p, with structural similarity to ATPase II, a candidate aminophospholipid translocase from bovine chromaffin granules. Loss of Dnf1p and Dnf2p virtually abolished ATP-dependent transport of NBD-labeled phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine from the outer to the inner plasma membrane leaflet, leaving transport of sphingolipid analogs unaffected. Labeling with trinitrobenzene sulfonic acid revealed that the amount of phosphatidylethanolamine exposed on the surface of Deltadnf1Deltadnf2 cells increased twofold relative to wild-type cells. Phosphatidylethanolamine exposure by Deltadnf1Deltadnf2 cells further increased upon removal of Drs2p, an ATPase II homolog in the yeast Golgi. These changes in lipid topology were accompanied by a cold-sensitive defect in the uptake of markers for bulk-phase and receptor-mediated endocytosis. Our findings demonstrate a requirement for Dnf1p and Dnf2p in lipid translocation across the yeast plasma membrane. Moreover, it appears that Dnf1p, Dnf2p and Drs2p each help regulate the transbilayer lipid arrangement in the plasma membrane, and that this regulation is critical for budding endocytic vesicles.

Melanization of Cryptococcus neoformans and Histoplasma capsulatum reduces their susceptibilities to amphotericin B and caspofungin.

The fungal pathogens Cryptococcus neoformans and Histoplasma capsulatum produce melanin-like pigments in the presence of L-dopa in vitro and during mammalian infection. We investigated whether melanization affected the susceptibilities of the fungi to amphotericin B, caspofungin, fluconazole, itraconazole, or flucytosine (5FC). Using the standard macrodilution MIC protocol (the M27A protocol) of the National Committee for Clinical Laboratory Standards for yeast, we found no difference in the susceptibilities of melanized and nonmelanized C. neoformans and H. capsulatum isolates. Killing assays demonstrated that melanization reduced the susceptibilities of both fungi to amphotericin B and caspofungin. Laccase-deficient C. neoformans cells grown with L-dopa were significantly more susceptible than congenic melanin-producing yeast to killing by amphotericin B or caspofungin. Preincubation of amphotericin B or caspofungin with melanins decreased their antifungal activities. Elemental analysis of melanins incubated with amphotericin B or caspofungin revealed an alteration in the C:N ratios of the melanins, which indicated binding of these drugs by the melanins. In contrast, incubation of fluconazole, itraconazole, or 5FC with melanins did not significantly affect the antifungal efficacies of the drugs or the chemical composition of the melanins. The results suggest a potential explanation for the inefficacy of caspofungin against C. neoformans in vivo, despite activity in vitro. Furthermore, the results indicate that fungal melanins protect C. neoformans and H. capsulatum from the activities of amphotericin B and caspofungin and that this protection is not demonstrable by standard broth macrodilution assays.

Potential application of plant lipid transfer proteins for drug delivery.

Ligand-binding proteins show an increasing interest as drug carriers and delivery systems [Wolf FA, Brett GM. Pharmacol Rev, 1000;52:207-36]. The wide binding properties of plant non-specific lipid transfer proteins such as LTP1 also offer many unexplored possibilities for such a task. In the present paper, by using intrinsic tyrosine LTP1 fluorescence, we survey, for the first time, the binding of wheat LTP1 with various ligands having cosmetic or pharmaceutical applications. LTP1 was found to bind skin lipids such as sphingosine, sphingomyelin, and cerebroside with an affinity of about one micromolar, low enough to allow a slow release of these molecules. Ether phospholipids and an azole derivative BD56 having antitumoral and/or antileishmania properties were also shown to bind LTP1 with similar affinity. Finally, amphotericin B, which is widely used as an antifungal drug, was shown to form a complex with LTP1, although no affinity could be determined. This binding study is a prerequisite for further work aimed at developing applications in LTP-mediated transport and controlled release of low molecular weight drugs.

Reduction of no synthase expression and tumor necrosis factor alpha production in macrophages by amphotericin B lipid carriers.

The present study compared the abilities of different lipid carriers of amphotericin B (AMB) to activate murine peritoneal macrophages, as assessed by their capacities to produce nitric oxide (NO) and tumor necrosis factor alpha (TNF-alpha). Although AMB alone did not induce NO production, synergy was observed with gamma interferon but not with lipopolysaccharide. This synergy could not be explained by the mobilization of the nuclear activation factor NF-kappaB by AMB. On the other hand, AMB induced TNF-alpha production without a costimulator and no synergy was observed. Anti-TNF-alpha antibodies did not influence NO production, and an inhibitor of NO synthase did not affect TNF-alpha production, indicating that the production of one of these effector molecules was independent of that of the other. The incorporation of AMB into lipid carriers reduced NO and TNF-alpha production with all formulations but more so with liposomes than with lipid complexes. NO production was correlated with the induction of NO synthase II, revealed by Western blotting. The extent of association of AMB with macrophages depended on the formulation, especially on the AMB/lipids ratio: the higher the ratio was, the greater the AMB association with macrophages. However, there was no clear correlation between AMB association with macrophages, whether internalized or bound to the membrane, and immunostimulating effects. These results may explain the reduced toxicities of lipid-based formulations of AMB.

Heat-induced superaggregation of amphotericin B modifies its interaction with serum proteins and lipoproteins and stimulation of TNF-alpha.

The purpose of the present study was to examine the influence of heat-induced superaggregation of Amphotericin B (AmB) in the Fungizone (FZ) formulation on its interaction with human serum components and relate this to reduced toxicity. Whole serum distribution studies showed that a significantly lower percentage of AmB from HFZ was recovered in the high-density lipoprotein (HDL), low-density lipoprotein (LDL), and triglyceride-rich lipoprotein (TRL) fractions and a greater percentage recovered in the lipoprotein-deficient plasma (LPDP), though the majority of both preparations were recovered in LPDP. Circular dichroism (CD) and difference absorption spectroscopy were used to determine the stability of FZ and heat-treated FZ (HFZ) in the presence of HDL, LDL, serum, and albumin. The CD studies indicate that the "core" aggregate of HFZ is more stable in the presence of HDL and LDL, whereas the FZ is less stable and more dynamic with the core aggregate dissociating to a greater extent in the presence of either purified lipoprotein. Absorption studies with whole serum and purified albumin suggest that FZ aggregates are far less stable in the presence of albumin than HFZ and that interaction with serum albumin is a dominant feature for both drug preparations. HFZ also has a different effect on the cytokine response in vitro. Studies using THP-1 human monocytes show that HFZ provokes a smaller release of tumor necrosis factor (TNF)-alpha than FZ. This cytokine may be associated with the unpleasant side effects of AmB. These findings suggest that heat-induced superaggregation of AmB alters its interaction with HDL, LDL, serum proteins, and monocytes, and these findings may be important in explaining the reduced toxicity of the superaggregated form of AmB.

Inhibitors of sterol biosynthesis and amphotericin B reduce the viability of pneumocystis carinii f. sp. carinii.

Pneumocystis carinii synthesizes sterols with a double bond at C-7 of the sterol nucleus and an alkyl group with one or two carbons at C-24 of the side chain. Also, some human-derived Pneumocystis carinii f. sp. hominis strains contain lanosterol derivatives with an alkyl group at C-24. These unique sterols have not been found in other pathogens of mammalian lungs. Thus, P. carinii may have important differences in its susceptibility to drugs known to block reactions in ergosterol biosynthesis in other fungi. In the present study, inhibitors of 3-hydroxy-3-methyglutaryl coenzyme A reductase, squalene synthase, squalene epoxidase, squalene epoxide-lanosterol cyclase, lanosterol demethylase, Delta(8) to Delta(7) isomerase, and S-adenosylmethionine:sterol methyltransferase were tested for their effects on P. carinii viability as determined by quantitation of cellular ATP levels in a population of organisms. Compounds within each category varied in inhibitory effect; the most effective included drugs targeted at squalene synthase, squalene epoxide-lanosterol cyclase, and Delta(8) to Delta(7) isomerase. Some drugs that are potent against ergosterol-synthesizing fungi had little effect against P. carinii, suggesting that substrates and/or enzymes in P. carinii sterol biosynthetic reactions are distinct. Amphotericin B is ineffective in clearing P. carinii infections at clinical doses; however, this drug apparently binds to sterols and causes permeability changes in P. carinii membranes, since it reduced cellular ATP levels in a dose-dependent fashion.