Physicochemical surrogates for in vitro toxicity assessment of liposomal amphotericin B.

Pharmaceutical toxicity evaluations often use in vitro systems involving primary cells, cell lines or red blood cells (RBCs). Cell-based analyses ('bioassays') can be cumbersome and typically rely on hard-to-standardize biological materials. Amphotericin B (AmB) toxicity evaluations are primarily based on potassium release from RBCs and share these limitations. This study evaluates the potential substitution of two physicochemical AmB toxicity approaches for the bioassay: Ultraviolet-visible spectroscopy (UV-vis) and in vitro drug release kinetics. UV-vis spectral analyses indicated that liposomal AmB's (L-AmB) main peak position (λmax) and peak ratio (OD346/OD322) are potential toxicity surrogates. Similarly, two first-order release parameters derived from USP-4 in vitro drug release analyses also provided linear relationships with toxicity. These were the initial, overall drug release rate and the ratio of loose to tight AmB pools. Positive slopes and high correlation coefficients (R2 > 0.9) characterized all interrelations between physicochemical parameters and toxicity. These tests converted the manufacturing variables' nonlinear (i.e., curvilinear) relationships with in vitro toxicity to linear responses. Three different toxicity attenuation approaches (2 manufacturing, 1 formulation), covering formulation composition and process aspects, support this approach's universality. These data suggest that one or more spectral and kinetic physicochemical tests can be surrogates for L-AmB in vitro toxicity testing.

Galleria mellonella for systemic assessment of anti-Candida auris using amphotericin B loaded in nanoemulsion.

Galleria mellonella is a model that uses adult larvae to assess the prophylactic, therapeutic, and acute toxic potential of substances. Given their benefits, G. mellonella models are being employed in investigations of systemic infections caused by highly resistant microorganisms. Among the multiresistant microorganisms, we highlight Candida auris, a yeast with high mortality potential and resistance. Among the potential drugs, amphotericin B (AmB) stands out; however, microbial resistance episodes and side effects caused by low selectivity have been observed. The incorporation of AmB into a nanoemulsion (NE) can contribute to the control of C. auris infections and resistance as well as decrease the side effects of this drug. This study aimed to develop AmB-loaded NE (NEA) and evaluate its antifungal action against C. auris in G. mellonella. NEs were obtained by using sunflower oil and cholesterol as the oily phase, polyoxyethylene 20 cetyl ether (Brij® 58) and soy phosphatidylcholine as the surfactant system, and PBS buffer as the aqueous phase. An alternative in vivo assay with G. mellonella for acute toxicity and infection was performed using adult stage larvae (200 mg to 400 mg). According to the obtained results, NE and NEA exhibited sizes of 43 and 48 nm, respectively. The PDI was 0.285 and 0.389 for NE and NEA, respectively. The ZP showed electronegativity for both systems, with -3.77 mV and -3.80 mV for NE and NEA, respectively. Acute toxicity showed that free AmB had greater acute toxicity potential than NEA. The survival assay showed high larval viability. NEA had a better antifungal profile against systemic infection in G. mellonella. It is concluded that the alternative model proved to be an efficient in vivo assay to determine the toxicity and evaluate the therapeutic property of free AmB and NEA in systemic infections caused by C. auris.

Analytical method development and comparability study for AmBisome® and generic Amphotericin B liposomal products.

Liposomal Amphotericin B, known as AmBisome®, is a life-saving antifungal product that sold $407 million in 2019. AmBisome® has a rather complex physical structure in that Amphotericin B (AmpB) forms a stable ionic complex with the lipid bilayer to maintain AmBisome®'s low toxicity and high stability in systemic circulation. Failed attempts to reproduce AmBisome®'s precise structure has resulted in faster drug release and higher toxicity both in vitro and in vivo. In this study, we established several analytical methodologies to quantify liposomal AmpB components, characterize thermal properties of the liposome, and determine particle size distribution, AmpB aggregation state, and drug release kinetics. We applied these methodologies together with in vitro hemolytic potential and antifungal activity tests to characterize multiple lots of AmBisome® and two generic products approved in India, Phosome® and Amphonex®. We also used Fungizone®, a micellar AmpB formulation, and "leaky" AmpB liposomes as negative controls. Our results showed that Phosome® and Amphonex® were both similar to AmBisome®, while Fungizone® and 'leaky" liposomes exhibited differences in both thermal properties and AmpB aggregation state, leading to faster drug release and higher toxicity. Due to the increased interest of the pharmaceutical industry in making generic AmBisome® and the lack of standard analytical methods to characterize liposomal AmpB products, the methodologies described here are valuable for the development of generic liposomal AmpB products.

Amphotericin B Loaded Nanostructured Lipid Carriers for Parenteral Delivery: Characterization, Antifungal and In vitro Toxicity Assessment.

BACKGROUND: Amphotericin B (AmB) is important for the treatment of systemic fungal infections. Nowadays, only intravenous administration (IV) of AmB has been available due to its low aqueous solubility. Two forms of AmB are available. The first is Fungizone®, a mixture of AmB and sodium deoxcycholate that produces severe nephrotoxicity. The second are lipid-based formulations that reduce nephrotoxicity, but they are costly and require higher dose than Fungizone®. Thus, a cheaper delivery system with reduced AmB toxicity is required. OBJECTIVE: To develop and characterize AmB loaded-nanostructured lipid carriers (AmB-loaded NLCs) for IV administration to reduce AmB toxicity. METHODS: AmB-loaded NLCs with different solid lipids were prepared by the high-pressure homogenization technique. Their physicochemical properties and the drug release profile were examined. The molecular structure of AmB, antifungal and hemolysis activities of developed AmB-loaded NLCs were also evaluated. RESULTS: AmB-loaded NLCs ~110 to ~140 nm in diameter were successfully produced with a zeta potential of ~-19 mV and entrapment efficiency of ~75%. In vitro release showed fast release characteristics. AmB-loaded NLCs could reduce the AmB molecular aggregation as evident from the absorbance ratio of the first to the fourth peak showing a partial aggregation of AmB. This result suggested that AmB-loaded NLCs could offer less nephrotoxicity compared to Fungizone®. In vitro antifungal activity of AmB-loaded NLCs showed a minimum inhibitory concentration of 0.25 µgmL-1. CONCLUSION: AmB-loaded NLCs present high potential carriers for effective IV treatment with prolonged circulation time and reduced toxicity.

In-vitro and in-vivo antileishmanial activity of inexpensive Amphotericin B formulations: Heated Amphotericin B and Amphotericin B-loaded microemulsion.

Amphotericin B (AmB) is effective against visceral leishmaniasis (VL), but the renal toxicity of the conventional form, mixed micelles with deoxycholate (M-AmB), is often dose-limiting, while the less toxic lipid-based formulations such as AmBisome® are very expensive. Two different strategies to improve the therapeutic index of AmB with inexpensive ingredients were evaluated on this work: (i) the heat treatment of the commercial formulation (H-AmB) and (ii) the preparation of an AmB-loaded microemulsion (ME-AmB). M-AmB was heated to 70 °C for 20 min. The resulting product was characterized by UV spectrophotometry and circular dichroism, showing super-aggregates formation. ME-AmB was prepared from phosphate buffer pH 7.4, Tween 80®, Lipoid S100® and Mygliol 812® with AmB at 5 mg/mL. The droplet size, measured by dynamic light scattering, was about 40 nm and transmission electron microscopy confirmed a spherical shape. Rheological analysis showed low viscosity and Newtonian behavior. All the formulations were active in vitro and in vivo against Leishmania donovani (LV9). A selectivity index (CC50 on RAW/IC50 on LV9) higher than 10 was observed for ME-AmB, H-AmB and AmBisome®. Furthermore, no important in vivo toxicity was observed for all the samples. The in-vivo efficacy of the formulations after IV administration was evaluated in Balb/C mice infected with LV9 (three doses of 1 mg/kg AmB) and no significant difference was observed between H-AmB, M-AmB, ME-AmB and AmBisome®. In conclusion, these two inexpensive alternative formulations for AmB showing good efficacy and selectivity for Leishmania donovani merit further investigation.

Assessment of the mutagenic, recombinogenic, and carcinogenic potential of amphotericin B in somatic cells of Drosophila melanogaster.

Amphotericin B (AmB) is an antifungal antibiotic extracted from Streptomyces nodosus. Its fungicidal activity depends primarily on its binding to the sterol group that is present in fungal membranes. In view of the toxicity of this drug, the purpose of this study was to evaluate its mutagenic, carcinogenic, and recombinogenic activity, based on the wing somatic mutation and recombination test (SMART) and the epithelial tumor detection test (wts) applied to Drosophila melanogaster. Larvae were chronically treated with different concentrations of AmB (0.01, 0.02, and 0.04 mg/mL). The results revealed that AmB is a promutagen exhibiting increase in the number of spots on individuals from high bioactivation (HB) cross with a high level of cytochrome P450. The results also indicate that the main genotoxic event induced by AmB is recombinogenicity. Homologous recombination can act as a determinant at different stages of carcinogenesis. For verification of carcinogenic potential of this compound, larvae from the wts/mwh and wts/ORR, flr3 were treated with the same three AmB concentrations used in the SMART assay. The results did not provide evidence that AmB has carcinogenic potential in wts/mwh individuals. However, individuals from wts/ORR, flr3 developed tumors at the highest concentration tested.

Assessment of in vitro antifungal efficacy and in vivo toxicity of Amphotericin B-loaded PLGA and PLGA-PEG blend nanoparticles.

Amphotericin B (AmB) is widely applied in treatment of systemic fungal infections. However, the emergence of severe adverse effects, such as nephrotoxicity, hepatotoxicity and hemolytic anemia, can limit its clinical use. Poly(lactide-co-glycolide) (PLGA) or poly(lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) blend nanoparticles containing AmB were developed with the aim to decrease AmB toxicity and propose the oral route for AmB delivery. Nanoparticles were characterized by particle size, polydispersity index, Fourier transform infrared spectroscopy, differential scanning calorimetry and X-ray diffraction analyses. The antifungal activity was evaluated against strains of Candida albicans and Cryptococcus neoformans. Toxicity was evaluated by hemolysis assay and after 7 days treatment in rats. Mean nanoparticle size was below 200nm with low polydispersity and AmB encapsulation efficiency higher than 90%. Nanoencapsulation resulted in AmB amorphization and no chemical interaction between drug and polymer. In C. albicans, minimum inhibitory concentration (MIC) of AmB-loaded PLGA-PEG nanoparticles was 2-fold higher than free AmB or marketed deoxycholate AmB (AmB-D), while MIC of AmB-loaded PLGA nanoparticles was 10-fold higher than AmB-loaded PLGA-PEG. In C. neoformans, the efficacy of AmB-loaded PLGA nanoparticles was comparable to free AmB, while AmB-loaded PLGA-PEG nanoparticles and AmB-D did not present MIC in tested concentration range. Nanoparticles inhibited the AmB-induced hemolysis. After 7 days treatment in rats, histologic examination revealed AmB-D treatment presented initial liver damage, while AmB-loaded nanoparticles did not present any hepatic cellular alteration. Kidney alteration was not observed in all treatments. Thus, PLGA and PLGA-PEG nanoparticles are promising carriers for AmB delivery with potential application in antifungal therapy.

Anfotericina B: uma revisão sobre suas diferentes formulações, efeitos adversos e toxicidade / Amphotericin B: a review on different formulations, side effects and toxicity

A incidência de infecções fúngicas invasivas tem aumentado, como consequência do contingente cada vez maior de pacientes com imunossupressão. O tratamento de infecções fúngicas com anfotericina B (AmB) está associado a efeitos adversos importantes, como nefrotoxicidade e toxicidade hematológica. Nesta revisão buscou-se abordar os estudos sobre AmB nas diferentes formulações, focando em suas características farmacológicas e toxicidade. Formulações lipídicas de AmB estão associadas a um risco menor de nefrotoxicidade, entretanto ainda há controvérsia sobre diferenças entre as duas formulações lipídicas de AmB disponíveis. Diferenças em relação ao perfil imunomodulatório e ligação a lipoproteínas podem explicar parte das diferenças clínicas existentes entre as formulações de AmB. A maioria dos estudos clínicos que avaliou a nefrotoxicidade associada à AmB em diferentes formulações não utilizou critérios validados para classificação do dano renal, o que dificulta sua comparação. A toxicidade hematológica relacionada ao uso de AmB é um fenômeno descrito desde os primórdios do seu uso clínico, entretanto poucos dados existem sobre sua frequência, fatores de risco e impacto nos desfechos clínicos. Dados precisos, e adequados ao contexto local, sobre a toxicidade de AmB nas suas diferentes formulações são necessários para uma adequada avaliação dos aspectos de farmacoeconomia e custo-efetividade...

Invasive fungal infections have emerged in recent years, as a consequence of increasing numbers of immunosuppressed patients. Treatment of these conditions with amphotericin B (AmB) has been associated with important side effects, such as nephrotoxicity and hematological toxicity. In this review we aimed to assess studies about different formulations of AmB, focusing on pharmacological properties and toxicity. Lipid formulations of AmB have been linked to a lower risk of nephrotoxicity; however, there is still controversy about differences between the two available lipid formulations. Differences in immunomodulatory profile and lipoprotein binding could partly explain clinical inequalities among AmB formulations. Most clinical trials that evaluated AmB-associated nephrotoxicity did not use validated criteria for renal injury classification, impairing comparability. Hematological toxicity associated with AmB treatments is an occurrence described since the beginning of its clinical use; nevertheless, few data exist about its frequency, risk factors, and clinical impact. Clear and more precise information, derived from local studies, is needed to an adequate evaluation about pharmacoeconomic aspects of AmB treatment and cost-effectiveness of lipid formulations...

The stress response resolution assay. II. Quantitative assessment of environmental agent/condition effects on cellular stress resolution outcomes in epithelium.

Cellular stress responses consist of a complex network of pathways and linked processes that, when perturbed, are postulated to have roles in the pathogenesis of various human diseases. To assess the impact of environmental insults upon this network, we developed a novel stress response resolution (SRR) assay for investigation of cellular stress resolution outcomes and the effects of environmental agents and conditions thereupon. SRR assay-based criteria identified three distinct groups of surviving cell clones, including those resembling parental cells, those showing Hprt/HPRT mutations, and a third type, "Phenotype-altered" clones, that occurred predominantly in cells pretreated with a chemical mutagen, was heterogeneous in nature, and expressed significant alterations in cell morphology and/or function compared with parental cells. Further evaluation of Phenotype-altered clones found evidence of various alterations that resembled epithelial-to-mesenchymal transition, phenotype switching, checkpoint dysfunction, senescence barrier bypass, and/or epigenetic reprogramming. Phenotype-altered clones were found to occur spontaneously in a cell line with a mutator phenotype, to represent the major surviving clone type in a variation of the SRR assay, and to be tumorigenic in nude mice. Assessment of SRR assay final results showed that pretreatment with a chemical mutagen induced significant changes in cellular stress response prosurvival capacity, in damage avoidance versus damage tolerance stress resolution outcomes, and in the damage burden in the final surviving cell populations. Taken together, these results support the conclusion that use of the SRR assay can provide novel insights into the role of environmental insults in the pathogenesis of cancer and other human diseases.

The stress response resolution assay. I. Quantitative assessment of environmental agent/condition effects on cellular stress resolution outcomes in epithelium.

The events or factors that lead from normal cell function to conditions and diseases such as aging or cancer reflect complex interactions between cells and their environment. Cellular stress responses, a group of processes involved in homeostasis and adaptation to environmental change, contribute to cell survival under stress and can be resolved with damage avoidance or damage tolerance outcomes. To investigate the impact of environmental agents/conditions upon cellular stress response outcomes in epithelium, a novel quantitative assay, the "stress response resolution" (SRR) assay, was developed. The SRR assay consists of pretreatment with a test agent or vehicle followed later by a calibrated stress conditions exposure step (here, using 6-thioguanine). Pilot studies conducted with a spontaneously-immortalized murine mammary epithelial cell line pretreated with vehicle or 20 µg N-ethyl-N-nitrososurea/ml medium for 1 hr, or two hTERT-immortalized human bronchial epithelial cell lines pretreated with vehicle or 100 µM zidovudine/lamivudine for 12 days, found minimal alterations in cell morphology, survival, or cell function through 2 weeks post-exposure. However, when these pretreatments were followed 2 weeks later by exposure to calibrated stress conditions of limited duration (for 4 days), significant alterations in stress resolution were observed in pretreated cells compared with vehicle-treated control cells, with decreased damage avoidance survival outcomes in all cell lines and increased damage tolerance outcomes in two of three cell lines. These pilot study results suggest that sub-cytotoxic pretreatments with chemical mutagens have long-term adverse impact upon the ability of cells to resolve subsequent exposure to environmental stressors.

Ventajas y desventajas de los distintos tipos de anfotericina en pediatría: revisión de la bibliografía / Advantages and drawbacks of amphotericin formulations in children: literature review

Las infecciones fúngicas han aumentado últimamente y el espectro de especies se ha modificado, en especial por el aumento del número de pacientes inmunocomprometidos. La anfotericina B es un antimicótico de amplio espectro; ha sido, durante los últimos 50 años, el fármaco de elección para el tratamiento antifúngico empírico y frente al aislamiento de hongos sensibles. Sin embargo, su uso se asocia con efectos adversos como fiebre, escalofríos, náuseas y vómitos (dependientes de la infusión) y, más significativamente, con nefrotoxicidad. Existen en la actualidad tres presentaciones farmacéuticas con lípidos que aumentan el índice terapéutico. A pesar de su uso extenso, existen aún interrogantes acerca de las dosis óptimas y costos que justifiquen su empleo en la edad pediátrica. No hay trabajos de alta calidad de evidencia sobre infecciones fúngicas documentadas en pediatría que comparen las cuatro presentaciones farmacéuticas disponibles. No obstante, pareciera que todas tienen similar eficacia. Los elevados costos de las anfotericinas asociadas a lípidos limitan su uso; sin embargo, la reducción de costos asociados a la menor nefrotoxicidad debería considerarsecuando se analizan los aspectos económicos de su indicación

Single-cell viability assessment with a novel spectro-imaging system.

Single-cell viability assessment by means of plural dye probes require the spectral and temporal analysis of microscopic images of the test cells. To meet this requirement, we have developed a simple and compact spectro-imaging system using an image slicer and a grism. The image slicer was made of a bundle of 100 optical fibers. The field of view is divided into 10 x 10 sections. The spectral data of each section could be recorded every 5 s in the range from 400 to 800 nm at 5 nm resolution. The viability changes of yeast or tobacco single-cells were measured with this system. Using BY-2 cells, for example, the response to a chemical stress of saponin was measured by means of two fluorescent probes. The spectral-spatial-temporal data of fluorescein and DNA bound ethidium bromide provided us with useful information about the dynamic change of cell membrane permeability from which the cell viability was assessed.

Carrier effects on biological activity of amphotericin B.

Amphotericin B (AmB), the drug of choice for the treatment of most systemic fungal infections, is marketed under the trademark Fungizone, as an AmB-deoxycholate complex suitable for intravenous administration. The association between AmB and deoxycholate is relatively weak; therefore, dissociation occurs in the blood. The drug itself interacts with both mammalian and fungal cell membranes to damage cells, but the greater susceptibility of fungal cells to its effects forms the basis for its clinical usefulness. The ability of the drug to form stable complexes with lipids has allowed the development of new formulations of AmB based on this property. Several lipid-based formulations of the drug which are more selective in damaging fungal or parasitic cells than mammalian cells and some of which also have a better therapeutic index than Fungizone have been developed. In vitro investigations have led to the conclusion that the increase in selectivity observed is due to the selective transfer of AmB from lipid complexes to fungal cells or to the higher thermodynamic stability of lipid formulations. Association with lipids modulates AmB binding to lipoproteins in vivo, thus influencing tissue distribution and toxicity. For example, lipid complexes of AmB can be internalized by macrophages, and the macrophages then serve as a reservoir for the drug. Furthermore, stable AmB-lipid complexes are much less toxic to the host than Fungizone and can therefore be administered in higher doses. Experimentally, the efficacy of AmB-lipid formulations compared with Fungizone depends on the animal model used. Improved therapeutic indices for AmB-lipid formations have been demonstrated in clinical trials, but the definitive trials leading to the selection of an optimal formulation and therapeutic regimen have not been done.