Amphotericin B Nephrotoxicity Research Articles

Functional Characterization of a Bacillus-Derived Novel Broad-Spectrum Antifungal Lipopeptide Variant against Candida tropicalis and Candida auris and Unravelling Its Mode of Action.

Limited treatment options, recalcitrance, and resistance to existing therapeutics encourage the discovery of novel antifungal leads for alternative therapeutics. Antifungal lipopeptides have emerged as potential candidates for developing new and alternative antifungal therapies. In our previous studies, we isolated and identified the lipopeptide variant AF4 and purified it to homogeneity via chromatography from the cell-free supernatant of Bacillus subtilis. AF4 was found to have broad-spectrum antifungal activity against more than 110 fungal isolates. In this study, we found that clinical isolates of Candida tropicalis and Candida auris exposed to AF4 exhibited low MICs of 4 to 8 mg/L. Time-kill assays indicated the in vitro pharmacodynamic potential of AF4. Biocompatibility assays demonstrated ~75% cell viability at 8 mg/L of AF4, indicating the lipopeptide's minimally cytotoxic nature. In lipopeptide-treated C. tropicalis and C. auris cells, scanning electron microscopy revealed damage to the cell surface, while confocal microscopy with acridine orange(AO)/propidium iodide (PI) and FUN-1 indicated permeabilization of the cell membrane, and DNA damage upon DAPI (4',6-diamidino-2-phenylindole) staining. These observations were corroborated using flow cytometry (FC) in which propidium iodide, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), and rhodamine 123 (Rh123) staining of cells treated with AF4 revealed loss of membrane integrity, increased reactive oxygen species (ROS) production, and mitochondrial membrane dysfunction, respectively. Membrane perturbation was also observed in the 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence study and the interaction with ergosterol was observed by an ergosterol binding assay. Decreased membrane dipole potential also indicated the probable binding of lipopeptide to the cell membrane. Collectively, these findings describe the mode of action of AF4 against fungal isolates by membrane disruption and ROS generation, demonstrating its antifungal potency. IMPORTANCE C. tropicalis is a major concern for candidiasis in India and C. auris has emerged as a resistant yeast causing difficult-to-treat infections. Currently, amphotericin B (AMB) and 5-flucytosine (5-FC) are the main therapeutics for systemic fungal infections; however, the nephrotoxicity of AMB and resistance to 5-FC is a serious concern. Antifungal lead molecules with low adverse effects are the need of the hour. In this study, we briefly describe the antifungal potential of the AF4 lipopeptide and its mode of action using microscopy, flow cytometry, and fluorescence-based assays. Our investigation reveals the basic mode of action of the investigated lipopeptide. This lipopeptide with broad-spectrum antifungal potency is apparently membrane-active, and there is a smaller chance that organisms exposed to such a compound will develop drug resistance. It could potentially act as a lead molecule for the development of an alternative antifungal agent to combat candidiasis.

Comparative Colloidal Stability of Commercial Amphotericin B Nanoformulations Using Dynamic and Static Multiple Light Scattering Techniques.

Amphotericin B (AmB) nanoformulations have been widely used for the treatment of invasive fungal infections in clinical practice, all of which are lyophilized solid dosage forms that improve storage stability. The colloidal stability of reconstituted lyophilized nanoparticles in an injection medium is a critical quality attribute that directly affects their safety and efficacy during clinical use. In the present study, the colloidal stability of commercial AmB nanoformulations, including AmB cholesteryl sulfate complex (AmB-CSC) and AmB liposome (AmB-Lipo), was evaluated using the dynamic (DLS) and static multiple light scattering (SMLS) techniques. Compared to the DLS technique, the SMLS technique allows for a more objective and accurate evaluation of the colloidal stability of AmB nanoformulations. The results obtained using the SMLS technique demonstrated that AmB-CSC and AmB-Lipo exhibited excellent colloidal stability in both sterile water and 5% dextrose injection. The disk-like structure of the AmB-CSC nanoparticles more readily adsorbed serum proteins to form protein corona compared to the spherical structure of AmB-Lipo after incubation with serum. Additionally, AmB-CSC and AmB-Lipo can significantly reduce the in vitro cytotoxicity and in vivo nephrotoxicity of AmB, which may be attributed to the good colloidal stability and the improved pharmacokinetic profiles of AmB nanoformulations. To the best of our knowledge, this study is the first to compare the colloidal stability of commercial AmB nanoformulations. These findings will provide useful information not only to inform the clinical use of available AmB nanoformulations but also for improving the design and conduct of translational research on novel AmB nanomedicines.

Turning weakness into strength: Albumin nanoparticle-redirected amphotericin B biodistribution for reducing nephrotoxicity and enhancing antifungal activity

As the gold standard treatment for invasive fungal infection, amphotericin B (AmB) is limited by its severe nephrotoxicity. It has been shown that AmB complex with albumin in vivo forms a sub-10 nm nanocomplex within kidney excretion size range and eventually induces the nephrotoxicity. This study presents an approach to take advantage of the “weakness” of such unique interaction between AmB and albumin to form AmB nanocomplex beyond the size range of kidney excretion. Herein, a novel strategy was developed by directly assembling molecular BSA into larger-sized nanostructures with the reconstructed intermolecular disulfide bond and hydrophobic interaction. The rich binding sites of AmB within BSA nanostructures enabled the efficient AmB loading and forming nanoparticle (AmB-NP) which exceeds the size range of kidney excretion (~ 60 nm). We found nanoassembly with BSA redirected biodistribution of AmB with a 2.8-fold reduction of drug accumulation in the kidney and significantly improved its renal impairment in mice. Furthermore, we found that nanoassembly with BSA significantly increased the biodistribution of AmB in brain and endowed it 100-folds increase in pharmacological effect against meningoencephalitis caused by common fungal pathogen Cryptococcus neoformans. Together, this study not merely overcomes the nephrotoxicity of AmB using its “weakness” by a nanoassembly method, and provides a new strategy for reducing toxicity of drugs with high albumin binding rate in vivo.

Comparison of intravenous sodium bicarbonate and sodium chloride combination versus intravenous sodium chloride hydration alone in reducing amphotericin B nephrotoxicity: a randomized clinical trial.

Background and purpose:The most important adverse reaction of amphotericin B (AmB) is nephrotoxicity. The aim of this study was to assess the potential effectiveness of intravenous saline + sodium bicarbonate versus intravenous sodium chloride hydration in preventing or attenuating AmB nephrotoxicity.Experimental approach:A randomized, non-placebo-controlled, single-blinded clinical trial was conducted in two adult hematology-oncology wards of Namazi hospital. Eligible patients were randomly assigned into either the normal saline or normal saline + sodium bicarbonate groups by the ratio of 1:2. In the normal saline group, 1000 mL of sodium chloride 0.9% (154 meq sodium) was given intravenously as two equal 500 mL volumes before and during the infusion of AmB. Patients in the saline + sodium bicarbonate group received 500 mL sodium chloride 0.9% (72 meq sodium) before and 500 mL isotonic sodium bicarbonate (72 meq sodium) intravenously during AmB infusion.Findings/Results:The rate of AmB nephrotoxicity was comparable between normal saline and sodium bicarbonate groups (54.2% and 41.6%, respectively; P = 0.3). This difference did not reach the level of statistical significance after considering AmB dose and duration of the treatment. The frequency of hypokalemia and hypomagnesemia did not differ significantly between the two groups even after adjusting the results according to AmB dose and treatment duration.Conclusion and implications:The results of the current preliminary clinical trial suggested that the combination of sodium bicarbonate and normal saline compared to normal saline alone appears to have no superiority in preventing or attenuating different studied aspects of AmB nephrotoxicity in patients with hematological malignancies.

Frequency and Associated Factors of Amphotericin B Nephrotoxicity in Hospitalized Patients in Hematology-Oncology Wards in the Southwest of Iran.

BackgroundNephrotoxicity is the most clinically significant adverse reaction of amphotericin B. Different aspects of amphotericin B (AmB) nephrotoxicity have not been studied well in our population.ObjectivesThe purpose of this study was to assess the frequency, time onset, and possible associated factors of AmB nephrotoxicity in hospitalized patients in hematology-oncology wards in the southwest of Iran.Patients and MethodsA cross-sectional, observational study was performed over a period of 9 months at 2 hematology-oncology and 1 hematopoietic stem cell transplantation wards at Namazi Hospital. Patients aged 15 years or older with no documented history of acute kidney injury or chronic kidney disease who were scheduled to receive formulations of AmB intravenously for at least 1 week were included. The required demographic and clinical data of the patients were recorded. Urine urea, creatinine, sodium, potassium, and magnesium levels were measured at days 0, 3, 5, 7, 10, and 14 of the AmB treatment. AmB nephrotoxicity based on serum creatinine increase, renal potassium wasting, hypokalemia, and hypomagnesemia were determined.ResultsAmong the 40 patients recruited for the study, 11 (27.5%) patients developed AmB nephrotoxicity with a mean ± standard deviation onset of 6.73 ± 2.36 days. In 5 patients, AmB nephrotoxicity resolved spontaneously without any intervention. According to the multivariate logistic regression model, none of the studied demographic, clinical, and paraclinical variables were significantly associated with AmB nephrotoxicity. The duration of hospitalization (P = 0.541) and the mortality rate (P = 0.723) were comparable between the patients with and without AmB nephrotoxicity. Hypokalemia and renal potassium wasting were identified in 45% and 27.5% of the patients during AmB treatment, respectively.ConclusionsNearly one-third (27.5%) of our cohort developed nephrotoxicity within the first week of AmB treatment. Hypokalemia and renal potassium wasting were more notable, affecting about one-half and one-third of the AmB recipients, respectively.

A double-blinded, placebo-controlled, multicenter clinical trial of N-acetylcysteine for preventing amphotericin B-induced nephrotoxicity

Objective: To evaluate the effectiveness of oral N-acetylcysteine (NAC) co-treatment in preventing amphotericin B (AmB)-induced nephrotoxicity (AIN), including creatinine clearance and biomarkers of renal function (cystatin C [Cys C] and kidney injury molecule-1 [KIM-1]).Methods: Either placebo or 600 mg oral NAC was given twice daily during the treatment course of AmB. Renal function test, serum as well as urinary level of Cys C and urinary KIM-1 were determined.Results: Among the study population (n = 54), 23 (42.59%) patients developed AmB nephrotoxicity during their treatment course. NAC co-treatment was significantly associated with mitigating AmB nephrotoxicity (OR = 0.286, 95% CI: 0.082 – 0.993; p = 0.049). No statistically significant difference regarding accuracy of measured biomarkers including serum creatinine, serum and urine Cys C and urine KIM-1 at days 0 and 7 of treatment in predicting and detecting AmB nephrotoxicity was identified. The changes in mean serum and urine Cys C and urine KIM during AmB treatment within and between treatment groups were not statistically significant.Conclusions: Co-treatment with 600 mg oral NAC twice a day during AmB treatment, after adjusting for multiple variables, was associated with prevention of AIN. However, significantly higher adverse reactions developed in the patients who were treated with NAC.

COMMUNICATION: Pharmacokinetics and Nephrotoxicity of Amphotericin B-Incorporated Poly(Ethylene Glycol)-Block-Poly(N-Hexyl Stearate l-aspartamide) Micelles

The purpose of this investigation was to study the pharmacokinetics and nephrotoxicity of amphotericin B (AmB), incorporated in poly(ethylene glycol)-block-poly(N-hexyl stearate l-aspartamide) (PEG-b-PHSA) micelles (AmB/PEG-b-PHSA). After AmB/PEG-b-PHSA or AmB for injection, United States Pharmacopeia (USP), was dosed intravenously in rats (0.8 mg/kg), serum was collected over 72 h, and organs collected at 72 h for AmB analysis. To test for the nephrotoxicity caused by AmB, renal markers of damage were assessed 24 h after a single injection of AmB/PEG-b-PHSA or AmB for injection, USP, focusing on detection of urinary enzymes. PEG-b-PHSA micelles caused a significantly lower area under serum concentration curve and higher clearance relative to AmB for injection, USP. PEG-b-PHSA micelles lowered the distribution of AmB in liver and lung tissues, but did not significantly lower the level of AmB in the kidneys relative to AmB for injection, USP. However, urine levels of N-acetyl-β-glucosaminidase and γ-glutamyltransferase were significantly lower for AmB/PEG-b-PHSA relative to AmB for injection, USP. In summary, PEG-b-PHSA micelles reduced the nephrotoxicity of AmB, the dose-limiting toxicity of this important antifungal agent.

Amphotericin B: side effects and toxicity

Amphotericin B (AmB) is a crucial agent in the management of serious systemic fungal infections. In spite of its proven track record, its well-known side effects and toxicity will sometimes require discontinuation of therapy despite a life-threatening systemic fungal infection. The mechanism of action of AmB is based on the binding of the AmB molecule to the fungal cell membrane ergosterol, producing an aggregate that creates a transmembrane channel, allowing the cytoplasmic contents to leak out, leading to cell death. Most of the efforts at improving AmB have been focused on the preparation of AmB with a lipid conjugate. AmB administration is limited by infusion-related toxicity, an effect postulated to result from proinflammatory cytokine production. The principal acute toxicity of AmB deoxycholate includes nausea, vomiting, rigors, fever, hypertension or hypotension, and hypoxia. Its principal chronic adverse effect is nephrotoxicity. AmB probably produces renal injury by a variety of mechanisms. Risk factors for AmB nephrotoxicity include male gender, higher average daily dose of AmB (≥35 mg/day), diuretic use, body weight ≥90 kg, concomitant use of nephrotoxic drugs, and abnormal baseline renal function. Clinical manifestations of AmB nephrotoxicity include renal insufficiency, hypokalemia, hypomagnesemia, metabolic academia, and polyuria due to nephrogenic diabetes insipidus. Human studies show convincingly that sodium loading in excess of the usual dietary intake notably reduces the incidence and severity of AmB-induced nephrotoxicity.

Amphotericin B-induced nephrotoxicity: characterization of blood and urinary biochemistry and renal morphology in mice

This study was conducted to characterize blood and urinary biochemistry, and renal morphology, after single or 1-week repeated dosing of mice with the polyene macrolide antibiotic, amphotericin B (AMB). AMB was intravenously administered to mice at 2 or 4 mg/kg for the single-dose experiment or once daily at 1 or 2 mg/kg for 1 week for the repeated-dose experiment. The most prominent histopathological findings included necrosis of the tubular epithelial cells in the thick ascending limb of Henle's loop in the renal outer medulla at a single dose of 2 or 4 mg/kg, and the severity of the lesion was dose-dependent. Blood chemistry and urinalysis revealed several changes suggestive of renal dysfunction such as reduction of plasma filtration ability (increases in plasma creatinine and blood urea nitrogen, a decrease in creatinine clearance) and polyuria accompanied with dehydration (decrease in renal water reabsorption, increases in plasma total protein and albumin) at a dose of 4 mg/kg in the single-dose experiment. Among the parameters analyzed, urinary lactate dehydrogenase was the most sensitive and reliable parameter for the prediction of AMB-induced nephrotoxicity in mice. These data provided comprehensive information on the nephrotoxicity of AMB and indicate useful markers for the sensitive detection of AMB-induced renal injury in mice.

Mixed micellar nanoparticle of amphotericin b and poly styrene-block-poly ethylene oxide reduces nephrotoxicity but retains antifungal activity

Mixed micellar nanoparticle consisting of amphotericin B (AmB) and poly styrene-block-poly ethylene oxide (PS-block-PEO) was prepared by high pressure homogenizer. Nephrotoxicity of the nanoparticle was investigated along with antifungal activity and self-aggregation status of the drug in the nanoparticle. Nephrotoxicity was markedly reduced when AmB was intravenously administered to rats as mixed micellar nanoparticle with PS-block-PEO in terms of transmission electron microscopy of tubular cells and creatinine clearance. Antifungal activity of AmB was not altered when the drug was in the form of mixed micellar nanoparticle compared to both conventional formulation and AmB micelle treated by same procedure without PS-block-PEO. Self-aggregation status of AmB molecules revealed monomeric in the mixed micellar nanoparticle with PS-block-PEO up to the therapeutic level of the drug (1-3 mM). The reduced nephrotoxicity of AmB in mixed micellar nanoparticle may be associated with the existence of the drug as monomeric form in the nanoparticle. Based on our result, formulation of AmB as mixed micellar nanoparticle with PS-block-PEO may be a promising alternative for the treatment of fungal diseases in patients who are at risk of renal dysfunction.

Synthesis of decapeptide ofl-aspartic acid and benzyl-l-aspartic acid by solid phase peptide synthesis

Polyene macrolide amphotericin B (AmB) is the drug of choice for the treatment of disseminated fungal infections. However, because of its pronounced side effects, the drug has limited applicability. There are few interesting reports, which state that co-administration of the drug with homo-peptide of polyaspartic acid reduces the side effects of the drug. In our present study, an approach has been made to systematically synthesize low molecular weight heteropeptides consisting of L-aspartic acid and its derivative. It was hypothesized that such heteropeptides will reduce the toxic side effects of the drug by facile hydrophobic binding between the polymer and the drug. We have employed the strategy of solid phase peptide synthesis (SPPS) to synthesize low molecular weight hetero-peptides by using L-aspartic acid and benzyl-L-aspartic acid to induce the hydrophobic binding between the peptide and the drug. In future, the proposed methodology can be employed to tailor other polypeptides substituted with benzyl groups to reduce the nephrotoxicity of AmB.

Potentiation of cisplatin and carboplatin cytotoxicity by amphotericin B in different human ovarian carcinoma and malignant peritoneal mesothelioma cells.

An in vitro study of the combined cytotoxicity of either cisplatin (CDDP) or carboplatin and amphotericin B (AmB) was undertaken on a set of different ovarian carcinoma (IGROVI, IGROVI-C10, OAW42) and peritoneal malignant mesothelioma (CFB-CARP1) cell lines and ascitic cells freshly obtained from ovarian cancer patients so as to investigate the possibility of overcoming their resistance to platinum compounds. Growth-inhibition curves obtained 6 days after a 2-h period of exposure to the drugs showed that AmB at 5-10 mg/l allowed a 5- to 10-fold decrease in the 50% growth-inhibitory concentrations (IC50) of CDDP and carboplatin on either sensitive or resistant cells. Intracellular platinum assays with IGROVI cells showed that AmB acted by increasing dramatically the platinum uptake at a proportion that accounted for the increase in cytotoxicity. In the subline IGROVI-C10, a 10-fold resistant subline of IGROVI, AmB at 10 mg/l allowed recovery to the level of sensitivity seen in the parental cell line in the absence of AmB but not to the level observed in the presence of AmB. Acquisition of resistance mechanisms that are independent of the regulation of platinum uptake might be involved in this cell line. Thus, AmB might act by increasing the intracellular concentration of platinum without modifying the resistance mechanism involved downstream. However, in our models an increase in the intracellular level of platinum was always sufficient for the recovery of chemosensitivity in vitro. We also show that the phosphodiesterase inhibiting methylxanthines act synergistically with AmB. The latter drugs are weakly toxic and could also attenuate the nephrotoxicity of AmB.

Urinary adenosine excretion in patients receiving amphotericin B

Background. Intravenous amphotericin B (AMB) administration in animals causes renal vasoconstriction, ischemia, and oliguria that may result in irreversible renal injury; the mechanism of AMB nephrotoxicity may be similar in human beings. Adenosine is excreted in urine by the ischemic kidney. We hypothesized that adenosine excretion and oliguria would be a marker for patients who later would manifest AMB-associated renal insufficiency and that pre-AMB saline administration (which ameliorates AMB nephrotoxidty) would negate the change in adenosine excretion and urine output. Methods. Twenty hospitalized patients being treated at the direction of their attending physician and who were receiving AMB (15 to 75 mg intravenously) had urine collected for 1 hour before and for 2 hours during AMB infusion. Eleven patients received normal saline solution (500 ml intravenously) before the AMB infusion; the other nine formed the comparator group. An aliquot of each urine collection was precipitated with perchloric acid to remove protein and cellular elements and centrifuged, and the supernatant was assayed for adenosine by using high-pressure liquid chromatography. Results. Infusion of AMB was associated with a decrease in mean urine output both in patients who received saline solution (245 before versus 149 ml/hr during AMB infusion, p = 0.04) and in patients in comparator group (139 versus 89 ml/hr, p = 0.027). The mean urinary adenosine excretion was unchanged in the saline-loaded group (0.1354 before versus 0.1255 mmol/hr during drug infusion, p = 0.25) and was decreased in the comparator group (0.2276 versus 0.1127 mmol/hr, p = 0.01). Development of renal insufficiency did not correlate with the change in urine output or adenosine excretion. Conclusions. AMB infusion in human beings results in decreased urine output and decreased adenosine excretion. The latter effect is prevented by a pre-AMB saline load. The changes in urine output and adenosine excretion are not predictive of the development of renal insufficiency.

Inhibiting Cholesterol Synthesis Reduces the Binding and Toxicity of Amphotericin B against Rabbit Renal Tubular Cells in Primary Culture

Renal tubular cells are a target of amphotericin B (AmB) toxicity, but the mechanisms involved in the tubular cell-AmB interactions are unknown. Ketoconazole was selected to lower the cholesterol content of rabbit renal tubular cells in primary culture. The consequences of cholesterol depletion on AmB nephrotoxicity was investigated in vitro as the inhibition of Na(+)-dependent phosphate uptake. After 1 h of exposure, AmB decreased phosphate uptake (49%, 77%, and 82% inhibition with 5, 10, and 20 microM of AmB, respectively). Pretreatment of cells with ketoconazole (10 microM for 24 h) reduced by 50% (P less than .01) the phosphate uptake inhibition induced by AmB, decreased cellular cholesterol synthesis (greater than 80% inhibition), and decreased AmB binding to cell membrane by 50%, as measured by the fluorescence extinction of a probe bound to tubular cell membrane. Incubation with exogenous exchangeable cholesterol again increased AmB binding to plasma membrane and restored AmB toxicity. These results demonstrate that the first step of AmB renal tubular toxicity is mediated by cellular cholesterol content and is parallel to the binding of AmB to cell membrane.

Amphotericin-B nephrotoxicity in humans decreased by sodium supplements with coadministration of ticarcillin or intravenous saline.

Previous observations suggest that salt loading can help reverse amphotericin-B induced nephrotoxicity. Evidence is presented indicating that sodium supplements provide prophylaxis against the development of amphotericin-B nephrotoxicity. In a retrospective study at Vanderbilt University, 14/21 patients receiving amphotericin B (target dose, 25 mg/day) without salt supplements developed impaired renal function; in 10 instances amphotericin B was temporarily withdrawn. In contrast, only 2/17 patients who received amphotericin B with ticarcillin (with its obligatory sodium supplement) developed nephrotoxicity (P less than 0.01). All four patients, who were receiving the combination of amphotericin B and ticarcillin and who had their ticarcillin therapy stopped, developed nephrotoxicity in the subsequent week. In a prospective observational study at Essen, 20 patients had 24 courses of amphotericin B (target dose, 40 mg/day) with routine supplementation of 1 liter of 0.9% sodium chloride daily. Only two patients showed evidence of nephrotoxicity and no dosage modification of amphotericin B was required in any patient. Four patients with initial evidence of mildly impaired renal function received full supplements without adverse effects or the development of nephrotoxicity. These observations suggest that routine parenteral administration of sodium supplements can help minimize the nephrotoxic potential of amphotericin B.