[Impurity profile analysis of amphotericin B using on-line two-dimensional high performance liquid chromatography-quadrupole time-of-flight mass spectrometry].

Amphotericin B (AmB) is a polyene-macrolide antimicrobial drug with a broad antibacterial spectrum and remarkable efficacy against deep fungal infections. It binds to ergosterol on the fungal cell membrane and alters its permeability, thereby destroying the membrane. AmB is a multicomponent antimicrobial medication that contains a wide range of impurities, rendering quality analysis extremely difficult. In the current Chinese Pharmacopoeia (Edition 2020) and European Pharmacopoeia (EP10.3), high performance liquid chromatography (HPLC) is applied to examine related substances in AmB. However, this technique presents a number of issues. For instance, the mobile phases used in the HPLC method described in both references contain nonvolatile inorganic salts, which cannot be coupled with a mass spectrometry (MS) detector. In addition, because the mobile phases used have a low pH, the component/impurities of AmB drug can easily be degraded or interconverted during the analytical process, leading to reduced analytical accuracy. Therefore, the accuracy and sensitivity of this method must be improved. In this study, a method based on on-line two-dimensional high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (2D HPLC-Q TOF/MS) was developed to analyze the impurity profile of AmB in accordance with the Chinese Pharmacopoeia (Edition 2020) and European Pharmacopoeia (EP10.3). The method combines on-line dilution and a multiple-capture HPLC system to achieve the efficient separation of AmB component/impurities. It also resolves the issue of poor solvent compatibility in 2D HPLC, increases the analytical flux, enhances the automation capability, reduces the mutual conversion of AmB and its impurities during the analytical process, and increases the detection sensitivity of the method. MS was also used to determine the structural inference of unstable components and impurities. An XBridge Shield C18 column (250 mm×4.6 mm, 3 µm) was used for first-dimensional-liquid chromatography with gradient elution using methanol-acetonitrile-4.2 g/L citric acid monohydrate solution (10∶30∶60, v/v/v, pH 4.7) as mobile phase A and methanol-acetonitrile-4.2 g/L citric acid monohydrate solution (12∶68∶20, v/v/v, pH 3.9) as mobile phase B. An Xtimate C8 column (10 mm×2.1 mm, 5 µm) was used as the trap column, and trapping and desalting were performed using 10 mmol/L ammonium formate aqueous solution containing 0.1% formic acid-acetonitrile (95∶5, v/v). An Xtimate C8 column (250 mm×2.1 mm, 5 µm) was used for second-dimensional-liquid chromatography with gradient elution using 10 mmol/L ammonium formate aqueous solution containing 0.1% formic acid-acetonitrile (95∶5, v/v) and 10 mmol/L ammonium formate aqueous solution containing 0.1% formic acid-acetonitrile (5∶95, v/v) as mobile phases. The data were collected in positive-ion mode. In this study, the structures of six impurities in amphotericin B were inferred, according to the fragmentation, the MS and MS2 spectra of each impurity. The developed method can be used to quickly and sensitively analyze the impurity profile of AmB. Furthermore, the research results on impurity profiles can be applied to guide improvements in AmB production.

Codetermination of antimicrobial agents in rabbit tear fluid using LC-MS/MS assay: Insights into ocular pharmacokinetic study.

Managing ocular microbial infections typically requires pharmacotherapy using antibiotic eye drops, such as moxifloxacin hydrochloride (MFX), combined with an antifungal agent like amphotericin B (AB). We carried out and validated an LC-MS/MS assay to quantify these compounds in rabbit tear fluid in order to look into the pharmacokinetics of these two drugs. We employed a protein precipitation technique for the extraction of drugs under examination. A Waters Symmetry C18 column was used to separate the analytes and internal standard. The composition of the mobile phase was like (A) 0.1% v/v formic acid in water and (B) methanol. The detection of MFX and AB was accomplished through the utilization of positive ion electrospray ionization under multiple reaction monitoring mode. The linearity curves for both analytes exhibited an acceptable trendline across a concentration range of 2.34-300 ng/mL for MFX and 7.81-1000 ng/mL for AB in surrogate rabbit tear fluid. The lower limit of quantitation for MFX was 2.34 ng/mL, while for AB, it was 7.81 ng/mL. The approach was strictly validated, encompassing tests of selectivity, linearity (with r2 > 0.99), precision, accuracy, matrix effects, and stability. Consequently, we employed this method to evaluate the pharmacokinetics profiles of MFX and AB in rabbit tear fluid following single topical doses.

Comparative metabolomics analysis of amphotericin B high-yield mechanism for metabolic engineering.

BACKGROUND: The polyene macrocyclic compound amphotericin B (AmB) is an important antifungal antibiotic for the clinical treatment of invasive fungal infections. To rationally guide the improvement of AmB production in the main producing strain Streptomyces nodosus, comparative metabolomics analysis was performed to investigate the intracellular metabolic changes in wild-type S. nodosus ZJB20140315 with low-yield AmB production and mutant S. nodosus ZJB2016050 with high-yield AmB production, the latter of which reached industrial criteria on a pilot scale. RESULTS: To investigate the relationship of intracellular metabolites, 7758 metabolites were identified in mutant S. nodosus and wildtype S. nodosus via LC-MS. Through analysis of metabolism, the level of 26 key metabolites that involved in carbon metabolism, fatty acids metabolism, amino acids metabolism, purine metabolism, folate biosynthesis and one carbon pool by folate were much higher in mutant S. nodosus. The enrichment of relevant metabolic pathways by gene overexpression strategy confirmed that one carbon pool by folate was the key metabolic pathway. Meanwhile, a recombinant strain with gene metH (methionine synthase) overexpressed showed 5.03 g/L AmB production within 120 h fermentation, which is 26.4% higher than that of the mutant strain. CONCLUSIONS: These results demonstrated that comparative metabolomics analysis was an effective approach for the improvement of AmB production and could be applied for other industrially or clinically important compounds as well.

Fatores de virulência e susceptibilidade a antifúngicos de Cryptococcus Spp / Virulence factors and susceptibility to Cryptococcus Spp. antifungals

Criptococose é uma doença grave que afeta tanto imunocomprometidos quanto imunocompetentes, com isso analisar a virulência é fundamental para novas terapêuticas. Objetivo: Analisar a capacidade de virulência e susceptibilidade aos antifúngicos de Cryptococcus spp. isolados de líquor de pacientes de hospital do norte do Paraná. Métodos: A partir de dois isolados clínicos C. neoformans e C. gattii, realizou-se a confirmação da identificação. Para a virulência, avaliou-se o tamanho da cápsula, capacidade de sobrevivência após exposição a neutrófilos, produção de melanina e urease. No antifungigrama por difusão em disco utilizou-se: anfotericina B, cetoconazol, voriconazol, itraconazol e miconazol. Resultados: C. gattii destaca-se por maior desenvolvimento da cápsula além da melhor capacidade de sobreviver a fagocitose em relação ao C. neoformans. No antifungigrama, ambos os isolados se apresentam sensíveis às drogas estudadas. Conclusão: Esses achados contribuem para a compreensão das diferentes patogêneses entre C. gattii e C. neoformans.

Cryptococcosis is a serious disease that can affect both immunocompromised and immunocompetent individuals, thus the virulence analysis is fundamental for the development of new treatments. Objective: To analyze the virulence and susceptibility of Cryptococcus spp. isolated from cerebrospinal fluid of patients from a hospital in the north of Paraná. Methods: From two clinical isolates, C. neoformans and C. gattii were confirmed and identified. For virulence, capsule size, survival capacity after exposure to neutrophils, melanin production and urease were evaluated. In the disc-diffusion method, the following antifungals were used: amphotericin B, ketoconazole, voriconazole, itraconazole and miconazole Results: It was observed that C. gattii presents greater results for development of the capsule beside presenting the best ability to survive phagocytosis in relation to C. neoformans. In the disc-diffusion method, both isolates presented sensitivity to the studied drugs. Conclusion: These findings contribute to the understanding of the different pathogens between C. gattii and C. neoformans.

Stability and Sterility of Extemporaneously Prepared Nonpreserved Cefazolin, Ceftazidime, Vancomycin, Amphotericin B, and Methylprednisolone Eye Drops.

PURPOSE: To determine in-use stability and sterility of fortified cefazolin, ceftazidime, vancomycin, amphotericin B, and methylprednisolone eye drops in a simulated inpatient setting with and without a mobile refrigerated container (MR). METHODS: Each drug was prepared and divided into 4 groups: 1) simulated patient use with the MR group: stored at 4°C and kept in the MR during drug administration, 2) simulated patient use without the MR (NoMR) group: stored at 4°C and no MR, 3) refrigerated control group: stored at 4°C, and 4) room temperature control group: stored at room temperature. Stability and sterility data were evaluated at days 0, 4, 7, 14, 21, and 28. Linear mixed-effects model and survival analysis were performed. RESULTS: Median time to 10% loss of concentration for in-use medications (MR/NoMR groups) was >28/27.9, 22.2/22.2, 19.4/19.4, 10.18/<4, and >28/>28 days for cefazolin, ceftazidime, vancomycin, amphotericin B, and methylprednisolone, respectively. There was no significant difference in the predicted concentration loss per day among all groups for vancomycin and methylprednisolone (all P > 0.05). For the other study medications, all room temperature control groups, the cefazolin NoMR group, and the ceftazidime NoMR group had significantly greater predicted concentration loss per day compared with the refrigerated control groups (all P ≤ 0.02). Culture results were negative for all drugs throughout the study. CONCLUSIONS: The NoMR group showed that the drug significantly degraded rapidly for cefazolin, ceftazidime, and amphotericin B. Implementation of MR could decrease the predicted loss of concentration per day for cefazolin and ceftazidime. In vitro antimicrobial activity and sterility were retained for 28 days.

Amphotericin-B and vancomycin-loaded chitosan nanofiber for antifungal and antibacterial application

In the present study, a mucoadhesive non-woven fiber mat (d= 116 nm) was fabricated by the electrospinning method using chitosan (80% Wt), polyethylene oxide (10% Wt), cysteine (4% Wt) and drugs (6% Wt), respectively. In addition, a comparative study was conducted to define effect of drugs and mucoadhesive agent on the nanofiber formation. FTIR, SEM, DSC and DMA were used to investigate the chemical and physical properties of the nanofibers. In vitro release of the drugs was assessed over a 48-hour period by the total immersion method. Release data were fitted to kinetic models, including the zero-order, first-order, Higuchi matrix, and Hixson-Crowell. Zone inhibition investigations were used to describe the inhibition content of vancomycin and amphotericin B loaded in the mats. The SEM images displayed a slight decrease in the fiber diameter with adding drugs and mucoadhesive agents. FTIR spectra confirmed that any undesirable reaction between VAN-AMB and CS-PEO was not observed. DSC test recognized the uniform distribution of drugs in the polymeric bead of the fiber without any crystal form. The elasticity modulus of the nanofiber was in an acceptable range for oral mucosa (approximately 5 Mpa). The results indicated that biodegradable mucoadhesive nanofibrous membranes released high concentrations of VAN in the first 24 hours, but the AMB release was affected in more controlled phenomena

A phase II study to evaluate the safety and efficacy of topical 3% amphotericin B cream (Anfoleish) for the treatment of uncomplicated cutaneous leishmaniasis in Colombia.

BACKGROUND: Pentavalent antimonials (Sb5) are the first-line drugs for treating cutaneous leishmaniasis in Colombia; however, given problems with toxicity, compliance, availability, and cost, it is imperative to look for better therapeutic options. Intravenous amphotericin B (AmB) has been used extensively to treat visceral leishmaniasis; however, evidence on its topical use for cutaneous leishmaniasis is limited. Anfoleish is a topical formulation based on 3% AmB, which was developed following GMP standards by HUMAX and PECET. Anfoleish was shown to be safe and efficacious in animal model and in an open label study in CL patients. Hereafter we show the results of the first controlled and randomized study assessing the safety and efficacy of Anfoleish administered topically, two or three times per day for 28 days, for the treatment of non-complicated cutaneous leishmaniasis in Colombia. METHODS: An open-label, randomized, non-comparative phase Ib/II clinical trial was performed. Adult volunteers with a parasitologically confirmed diagnosis of cutaneous leishmaniasis were randomly allocated to receive Anfoleish cream either 3 (TID group) or 2 (BID group) times per day for 4 weeks. RESULTS: 80 out of 105 subjects screened were included in the study. In intention to treat analysis, final cure was observed in 13 (32.5%) out of 40 subjects (IC 95% = 20.1-48) and in 12 (30%) out of 40 subjects (IC 95% = 18.1-45.5) in the BID and TID group respectively. In the per protocol analysis, cure rates were 39.4% (n = 13) (IC 95% = 24.7-56.3) and 35.3% (n = 12) (IC 95% = 21.5-52.1) in the BID and TID groups respectively. Anfoleish proved to be safe, and the few adverse events reported were local, around the area of application of the cream, and of mild intensity. CONCLUSION: Anfoleish showed to be a safe and well-tolerated intervention. Its efficacy results however do not support at this time continuing with its clinical development or recommending it for the treatment of CL. Additional, studies to improve its current formulation are needed before thinking in conducting additional studies in patients. TRIAL REGISTRATION: Registered in clinicaltrials.gov NCT01845727.

Amphotericin B concentrations in healthy mallard ducks (Anas platyrhynchos) following a single intratracheal dose of liposomal amphotericin B using an atomizer.

Aspergillosis is a fungal infection that primarily affects the respiratory tract. Amphotericin B has broad antifungal activity and is commonly used to treat aspergillosis, a fungal pneumonia that is a common sequela in oiled waterfowl as well as other birds in wildlife rehabilitation. Pharmacokinetic parameters of nebulized amphotericin B in an avian model have been reported, but those of direct intratracheal delivery have yet to be established. The objective of this study was to evaluate if a single 3 mg/kg dose of liposomal amphotericin B delivered intratracheally using a commercial atomizer would achieve plasma and lung tissue concentrations exceeding targeted minimum inhibitory concentrations (MIC) for Aspergillus species in adult mallard ducks (Anas platyrhynchos). Following intratracheal delivery, amphotericin B was present in lung parenchyma at concentrations above the targeted MIC of 1 µg/g for up to 9 days post-administration; however, distribution of the drug was uneven, with the majority of the drug concentrated in one lung lobe. Concentrations in the contralateral lung lobe and the kidneys were above the targeted MIC 1 day after administration but declined exponentially with a half-life of approximately 2 days. Plasma concentrations were never above the targeted MIC. Histological examination of the trachea, bronchi, lungs, heart, liver, and kidneys did not reveal any toxic changes. Using a commercial atomizer, intratracheal delivery of amphotericin B at 3 mg/kg resulted in lung parenchyma concentrations above 1 µg/ml with no discernable systemic effects. Further studies to establish a system of drug delivery to both sides of the pulmonary parenchyma need to be performed, and the efficacy of this treatment for disease prevention remains to be determined.

Utilizing online-dual-SPE-LC with HRMS for the simultaneous quantification of amphotericin B, fluconazole, and fluorocytosine in human plasma and cerebrospinal fluid.

Amphotericin B (AMB), fluconazole (FZ), and fluorocytosine (FC) are recommended for HIV-associated cryptococcal meningitis (CM) patients as preferred antibiotics. This study presents a fast and automated online-dual-solid phase extraction (SPE)-LC coupled with high resolution mass spectrometer (HRMS) method to simultaneously measure the concentrations of AMB, FZ, and FC in human plasma and cerebrospinal fluid (CSF). Automated sample clean-up was performed on the human plasma and CSF samples with stop-flow heart-cutting two dimensional (2D) separation using a online-dual-SPE system, allowing retention and accumulation of AMB, FZ, and carbamazepine (CBZ, Internal standard (IS)) by the Oasis®HLB cartridge, and retention and accumulation of FC and 5-methylcytosine hydrochloride (MC, IS) by the HyperSep Hypercarb cartridge respectively. Followed by LC elution, quantification by Q-Exactive Hybrid Quadrupole-Orbitrap with targeted-selected ion monitoring (t-SIM) mode was applied to simultaneously determine the concentrations of AMB, FZ and FC. The bioanalysis was achieved in a total running time of 7min. The method was fully validated according to FDA guidelines. The lowest limit of quantification (LLOQ) was 0.04, 0.04, and 0.40µgmL-1 for AMB, FZ, and FC, respectively. AMB, FZ, and FC levels were linear in the ranges of 0.04-2.00µgmL-1, 0.04-2.00µgmL-1 and 0.40-20.00µgmL-1, respectively. The method showed good performance for human plasma and CSF samples with linearity (R2>0.99), intra-day and inter-day precision (relative standard deviation, RSD<4.32% and <4.06%, respectively), recovery (89.93-93.28% and 90.09-93.58%, respectively) and matrix effect (96.35-103.78% and 92.32-101.48%, respectively). The validated method was successfully applied in real samples of Chinese patients. Overall, our results indicate that this fully automated, sensitive, and reliable online-dual-SPE-LC-HRMS method is effective for therapeutic drug monitoring (TDM) of AMB, FZ, and FC levels.

Epidemiología, factores de riesgo y sensibilidad in vitro en candidemias por especies diferentes de Candida albicans / Epidemiology, risk factors and in vitro susceptibility in candidaemia due to non-Candida albicans species

Antecedentes. La infección fúngica invasora ha aumentado en los últimos años por el incremento de los factores de riesgo; la candidemia es la principal manifestación clínica. Candida albicans es la especie más frecuente, aunque actualmente se ha observado un aumento en otras especies del género. Objetivos. Analizar la epidemiología, los factores de riesgo y la sensibilidad antifúngica de los aislamientos en hemocultivos de especies de Candida diferentes de C.albicans en nuestro hospital en los últimos 12años. Métodos. Se estudiaron retrospectivamente las características epidemiológicas de 107 pacientes con candidemia ingresados en nuestro hospital. Se determinó la sensibilidad de las especies de Candida al fluconazol, el itraconazol, el voriconazol, la anfotericinaB, la 5-fluorocitosina, la caspofungina, la micafungina y la anidulafungina mediante el método de microdilución Sensititre Yeast One (Izasa, España). Resultados. De los 109 aislamientos, 59 correspondieron a las siguientes especies de Candida (diferentes de C.albicans): 25 Candida parapsilosis complex, 14 Candida glabrata complex, 13 Candida tropicalis, 4 Candida krusei, una Candida lipolytica, una Candida membranaefaciens y una Candida pulcherrima. El factor de riesgo más frecuente en adultos y niños con candidemias por estas especies fue ser portador de catéter. El 8,5% de estos aislamientos fueron resistentes al fluconazol. Conclusiones. El resultado de nuestro trabajo confirma la necesidad de conocer la epidemiología de las especies de Candida diferentes de C.albicans, su sensibilidad in vitro y los factores de riesgo asociados, especialmente en pacientes con dichos factores (AU)

Background. Invasive fungal infection (IFI) has increased in recent years due to there being a greater number of risk factors. IFI caused by Candida is the most frequent, and although Candida albicans is the most isolated species, there is currently a decrease of C. albicans and an increase of other species of the genus. Aims. To analyse the epidemiology, risk factors, and antifungal susceptibility of blood culture isolates of non-C.albicans Candida species in our hospital in the last 12years. Methods. A retrospective study was conducted on 107 patients with candidaemia admitted to our hospital. Candida isolates susceptibility to fluconazole, itraconazole, voriconazole, amphotericinB, 5-fluorocytosine, caspofungin, micafungin, and anidulafungin was determined by means of a microdilution technique (Sensititre Yeast One; Izasa, Spain). Results. From a total of 109 strains, 59 belonged to non-C. albicans Candida species: 25 Candida parapsilosis complex, 14 Candida glabrata complex, 13 Candida tropicalis, 4 Candida krusei, 1 Candida lipolytica, 1 Candida membranaefaciens, and 1 Candida pulcherrima. The most common risk factor in adults and children was catheter use. It was observed that 8.5% of those non-C.albicans strains were resistant to fluconazole. Conclusions. The results of this work confirm that it is necessary to know the epidemiology of non-C.albicans Candida species, the in vitro susceptibility of the species involved, and the main risk factors, especially in patients with predisposing conditions (AU)

Interference of melanin in the susceptibility profile of Sporothrix species to amphotericin B / Interferencia de la melanina en la sensibilidad de especies del género Sporothrix a la anfotericina B

Background. The presence of melanin in the fungal cell is a major virulence factor of the genus Sporothrix since it protects the fungal cells against the defense systems. Aims. The present study aimed to investigate the interference of melanin in the susceptibility of Sporothrix brasiliensis and Sporothrix schenckii sensu stricto to amphotericin B and itraconazole, drugs recommended as therapy for disseminated and subcutaneous sporotrichosis, respectively. Methods. Yeast cells were cultivated in minimal medium with or without l-DOPA in order to induce the production of melanin. Microdilution and killing assay methods were used to determine the antifungal activity against yeast cells with different mounts of melanin. Results. The killing assay showed that melanization protected isolates within the S. schenckii complex from amphotericin B, particularly in the lower concentrations tested. Conclusions. Studies combining amphotericin B and inhibitors of melanin are required in order to avoid this effect (AU)

Antecedentes. La presencia de melanina en la célula fúngica es un importante factor de virulencia del género Sporothrix en el proceso de infección, pues protege al hongo de la acción del sistema inmunitario. Objetivos. El objetivo del presente estudio fue investigar la interferencia de la melanina en la sensibilidad de Sporothrix brasiliensis y Sporothrix schenckii sensu stricto a la anfotericina B y el itraconazol, antifúngicos recomendados como terapia para la esporotricosis diseminada y la subcutánea, respectivamente. Métodos. Se cultivaron células en la fase levaduriforme en medio mínimo con o sin l-DOPA con el fin de inducir la producción de melanina. La valoración de la actividad antifúngica sobre células de Sporothrix con diferente contenido en melanina se realizó mediante microdilución en caldo y curvas de mortalidad. Resultados. El ensayo mostró que la melanización protege de la acción de la anfotericina B a las especies del complejo S. schenckii, particularmente en las concentraciones más bajas ensayadas. Conclusiones. Se requieren estudios de combinación entre diferentes clases de antifúngicos o de anfotericina B con inhibidores de la melanina, con el fin de disminuir o evitar este efecto (AU)

Purity determination of amphotericin B, colistin sulfate and tobramycin sulfate in a hydrophilic suspension by HPLC.

A suspension comprising of the three antibiotic substances amphotericin B, colistin sulfate and tobramycin sulfate is often used in clinical practice for the selective decontamination of the digestive tract of patients in intensive care. Since no detailed procedures, specifications or stability data are available for manufacturing this suspension, there may be discrepancies regarding formulation and stability of suspensions prepared in different pharmacies. The aim of this work is to develop a standardized formulation and to determine its stability under defined storage conditions. This would help guarantee that all patients receive the same preparation, therefore ensuring similar efficacy and improved safety. The first step in this process is to develop the required analytical tools to measure the content and purity of the drug substances in this complex mixture. In this paper, the development and validation of these tools as well as the development of the drug suspension formulation is described. The formulation comprises of Ampho-Moronal(®)-Suspension (Dermapharm) and a buffered, preservated aqueous solution of colistin sulfate and tobramycin sulfate. Two simple, well established high-performance liquid chromatography (HPLC) methods in the European Pharmacopoeia (EP) for impurity profiling of the two active ingredients amphotericin B and colistin sulfate were combined with a newly developed sample extraction procedure for the suspension. Sufficient selectivity and stability-indicating power have been demonstrated. Additionally, a new robust routine method was developed to determine possible degradation products of tobramycin sulfate in the investigated suspension. The specificity, precision, accuracy and linearity of the analytical procedures were demonstrated. The recovery rate was in the range of 90-110%. The precision results for the calculated impurities showed variation coefficients of <10%. The calibration curves were found to be linear with correlation of greater than 0.9994 for all components. The results show the suitability of the methods for the quality control analysis of the suspension.

Long-term sustained-released in situ gels of a water-insoluble drug amphotericin B for mycotic arthritis intra-articular administration: preparation, in vitro and in vivo evaluation.

Amphotericin B (AMB) was often used in intra-articular injection administration for fungal arthritis, because it could often bring a satisfactory therapeutic efficacy and a minimum systemic toxic side effect. However, because of the multiple operations and the frequent injections, the compliance of the patients was bad. Therefore, to develop a long-term sustained-released preparation of AMB for mycotic arthritis intra-articular administration is of great significance. The purpose of present study was to develop a long-term sustained-released in situ gel of a water-insoluble drug AMB for mycotic arthritis intra-articular administration. Based on the evaluations of the in vitro properties of the formulations, the formulation containing 10% (w/w) ethanol, 15% (w/w) PG, 0.75% (w/w) HA, 5% (w/w) purified soybean oil, 0.03% (w/w) α-tocopherol, 15% (w/w) water and 55% (w/w) glyceryl monooleate was selected as a suitable intra-articular injectable in situ gel drug delivery system for water-insoluble drug AMB. Furthermore, the results of the in vivo study on rabbits showed that the selected formulation was a safe and effective long-term sustained-released intra-articular injectable AMB preparation. Therefore, the presented in situ AMB gel could reduce the frequency of the administration in the AMB treatment of fungal arthritis, and then would get a good patient compliance.

Análisis de la utilización de anfotericina B liposomal / Analysis of the use of liposomal amphotericin B

Antecedentes. El aumento en los últimos años de pacientes inmunodeprimidos y el uso de fármacos citotóxicos e inmunosupresores ha aumentado la incidencia de infecciones fúngicas invasivas. Objetivos. Evaluar la utilización de anfotericina B liposomal de acuerdo con la indicación, la dosis, la efectividad y la nefrotoxicidad. Métodos. Estudio retrospectivo de un período de 8 años donde se tomaron como referencia las indicaciones y posología descritas en la ficha técnica. La efectividad se midió en función de la resolución de la infección, no recurrencia o aparición, supervivencia a los 7 días, no suspensión y no adición de otro antifúngico. La efectividad se analizó en función de indicación, dosis, duración del tratamiento, dosis acumulada y comorbilidad. La nefrotoxicidad fue medida como un aumento de la creatinina sérica por encima de 2 veces la basal. Resultados. Se trataron 47 episodios, de los cuales el 91,5% de los tratamientos se ajustaron a las indicaciones. Se logró efectividad en un 44,7% de los casos: 33% en neutropénicos, 50% de las aspergilosis, 60% de las candidiasis y 100% de las leishmaniasis. Las tasas de respuesta en los tratados durante ≤ 15 días y > 15 días fueron de 25 y 56,5% (p = 0,039), respectivamente. Las causas principales del fracaso fueron exitus (23%) y falta de efectividad (17%). Un 9% de los pacientes sufrió nefrotoxicidad. Conclusiones. Los datos de efectividad y nefrotoxicidad concuerdan con los obtenidos en otros estudios. Su efectividad se ha visto asociada con la duración del tratamiento. La anfotericina B liposomal es un fármaco seguro con efectividad moderada (AU)

Background. The increase in immunosuppressed patients and in the use of cytotoxic and immunosuppressive agents in the last few years has led to a rise in the incidence of invasive fungal infections. Aims. he objective of this study is to evaluate the use of liposomal amphotericin B according to its indication, dosage, effectiveness and nephrotoxicity. Methods. This is a retrospective study over a 8 year-period. Indications and dosage described in the Summary of Product Characteristics were taken as reference. Effectiveness was measured in terms of resolution of infection, no recurrence or emergence, survival at 7th day, no discontinuation and no addition of another antifungal. Effectiveness was also analysed in relation to indication, dosage, treatment duration, cumulative dose and comorbidity. Nephrotoxicity was defined as a doubled serum creatinine when compared with basal values. Results. A total of 47 episodes were analysed, with 91.5% of treatments being adequate for the indications. Effectiveness was achieved in 44.7% of cases: 33% in neutropenic patients, 50% of aspergillosis, 60% of candidiasis, and 100% of the leishmaniasis. Response rates in patients treated for 15 days or less and those for more than 15 days were 25 and 56.5% (P = .039), respectively. The main causes of failure were death (23%) and lack of effectiveness (17%), with 9% of patients suffering from nephrotoxicity. Conclusions. The effectiveness and nephrotoxicity data agree with those obtained in other studies. Liposomal amphotericin B is a safe drug with a moderate effectiveness that is associated with the duration of the treatment (AU)

Determination of amphotericin B in PLA-PEG blend nanoparticles by HPLC-PDA

In this work, we developed and validated an effective reversed-phase HPLC method with photodiode array (PDA) detection for the quantitative analysis of amphotericin B (AmB) in poly(lactide)-poly(ethylene glycol) (PLA-PEG) blend nanoparticles. Chromatographic runs were performed on a reverse phase C18 column using a mobile phase comprising a 9% acetic acid and acetonitrile mixture (40:60, v/v) under isocratic elution with a flow rate of 1 mL/min. AmB was detected at a wavelength of 408 nm. The validation process was performed considering the selectivity, linearity, precision, accuracy, robustness, limit of detection (LOD) and limit of quantitation (LOQ) of the method. A concentration range of 1-20 µg/mL was used to construct a linear calibration curve. The LOQ and LOD were 55 and 18 ng/mL, respectively. The mean recovery of AmB from the samples was 99.92% (relative standard deviation (RSD) = 0.34%, n=9), and the method was robust for changes in the flow rate of the mobile phase (maximum RSD=4.82%). The intra- and inter-assay coefficients of variation were less than 0.59%. The method was successfully used to determine the entrapment efficiency of AmB in PLA-PEG blend nanoparticles.

Neste trabalho desenvolveu-se e validou-se um efetivo método por cromatografia líquida de alta eficiência (CLAE) em fase reversa com detecção por fotodiodos para a análise quantitativa de anfotericina B (AmB) em nanopartículas compostas por blendas de poli(ácido lático)-polietilenoglicol (PLA-PEG). Corridas cromatográficas foram realizadas sob coluna C18 de fase reversa com fase móvel consistindo de ácido acético 9% e acetonitrila (40:60, v/v), em eluição isocrática com fluxo de 1 mL/min. A AmB foi detectada no comprimento de onda de 408 nm. O processo de validação foi realizado considerando a seletividade, linearidade, precisão, exatidão, robustez, limite de detecção (LD) e limite de quantificação (LQ) do método. Uma faixa de concentração entre 1-20 µg/mL foi usada para obter a curva-padrão linear. Os valores de LD e LQ foram 55 e 18 ng/mL, respectivamente. A recuperação média da AmB a partir das amostras foi de 99,92% (desvio padrão relativo = 0,34%, n=9) e o método foi robusto, considerando alterações no fluxo da fase móvel (desvio padrão relativo máximo=4,82%). Os coeficientes de variação intra e inter dia foram inferiores a 0,59%. O método foi utilizado com sucesso para a determinação da eficiência de encapsulação da AmB em nanopartículas de PLA-PEG.

Leishmaniasis visceral, nuestra experiencia en 30 años / Visceral leishmaniasis, our experience in 30 years

Objetivos. El kala azar o leishmaniasis viscera (LV) es una de las enfermedades más olvidadas del mundo. En los países en desarrollo, 350 millones de personas están en riesgo de contraer LV, habiendo 2 millones de casos nuevos al año, En España, a partir de 1982, se hizo enfermedad de declaración obligatoria, notificándose unos 90 casos/año. A partir de 1995 pasó a ser enfermedad de declaración obligatoria en zonas endémicas, considerándose Aragón una de ellas. Pacientes y métodos. Estudio descriptivo retrospectivo de 14 casos de LV confirmados en edad pediátrica (0 a 14 años) ingresados en la Unidad de Infecciosos del Hospital Infantil Miguel Servet, de Zaragoza, entre enero de 1980 a diciembre de 2010. Resultados. Fueron ingresados 16 niños con el diagnóstico de LV. El estudio se lleva a cabo con datos de 14 pacientes. La edad media de presentación fue 3 años (rango 9 meses-12 años, mediana 19 meses). El 64% de los pacientes procedían del medio rural. Al ingreso presentaban una media de 3.700 leucocitos, con neurtropenia en el 71% de los pacientes. Existían trombopenia (<150.000 plaquetas/mm3) en un 77% de los casos. Presentaban anemia el 100% de los pacientes con una media de hemoglobina de 7,7 mg/dl. Un 64 % de los niños debutaron con pancitopenia. La VSG se elevó por encima de 20 mm/h en el 100% de los casos. En todos los pacientes se realizó aspirado de médula ósea, visualizándose el parásito en todas las muestras, confirmando el diagnóstico de LV. Todos los casos fueron tratados con meglumina antimoniato, excepto el último registrado en 2010 que recibió anfotericina B liposomal, todos con buena respuesta clínica (...) (AU)

Objectives. Kala azar o viscera leishmaniasis (LV) is one of the most neglected disease in the world. In developing countries, 350 million people are at risk of having LV and 2 million cases are reported every year. In Spain, since 1982 LV has become a compulsory notifiable disease with around 90 cases reported every year. In 1995, it was declared notifiable disease in endemic areas and one of them was Aragón. Patients and Methods. Retrospective study of 14 VL cases. All of them in patients between 0-14 years old admitted into the Infectious Diseases Unit at Children´s Hospital Miguel Servet in Zaragoza between January 1980 to December 2010. Results. A total of 16 children were admitted with the diagnosis of I. V. The study was carried out with data of 14 of these patients. The mean age at diagnosis was 3 years (range 9 months-12 years, median 19 months). The 64% of them came from rural areas. At admission, they had 3700 leukocyte average with neutropenia in 71% of those cases. All of them (100%9 were anemic with a 7.7 mg/ld hemoglobin/dl range but only nearly 77% has thrombocytopenia (<150,000 platelets/mm3). The 64% of the children debuted with pancytopenia. ESR rose were more than 20 mm/h in all cases (...) (AU)

Treatment of invasive fungal infections in high risk hematological patients. The outcome with liposomal amphotericin B is not negatively affected by prior administration of mold-active azoles

Existe cierta inquietud sobre una reducción del efecto de la anfotericina B liposomal (L-AmB) administrada secuencialmente después de la administración de azoles activos frente a mohos debido a un posible antagonismo en su mecanismo antifúngico. Para investigar este posible efecto en la práctica clínica, hemos estudiado retrospectivamente 182 pacientes hematológicos con infecciones fúngicas invasivas (IFI) de alto riesgo que fueron tratados con L- AmB. En total, 96 pacientes (52,7%) tenían IFI posible, 52 (28,6%) probable y 34 (18,7%) probada de acuerdo con la clasificación de la EORTC. La mayoría presentaban aspergilosis invasiva. Comparamos los pacientes con exposición previa a azoles activos frente a mohos (n=100) con aquellos no expuestos (n=82). El grupo con exposición previa a azoles activos frente a mohos incluía más pacientes con características de alto riesgo de IFI, como leucemia mieloide aguda (p<0,05) y neutropenia prolongada (p<0,05). Se alcanzó una respuesta favorable en la IFI, definida como una respuesta completa o parcial, en 75,0% y 74,4% de los pacientes de la cohorte completa y en 66,0% y 74,4% de los pacientes con IFI probable o probada en los dos grupos. Ninguna de estas diferencias fue significativa. El análisis multivariante mostró que la enfermedad basal y la disfunción renal eran factores adversos para la respuesta en la IFI (p<0,05). La supervivencia fue peor en los pacientes tratados con azoles de amplio espectro (p<0,05) y en aquellos en los que no se resolvió la neutropenia (p<0,05). En conclusión, la eficacia del tratamiento con LAmB de una infección fúngica de brecha probablemente no se vea afectada por la exposición previa a un tratamiento profiláctico con azoles activos frente a mohos, dependiendo la supervivencia más bien de los factores del huésped y de la enfermedad de base(AU)

There are concerns of a reduced effect of liposomal amphotericin B (L-AmB) given sequentially after mold-active azoles due to a possible antagonism in their antifungal mechanism. To investigate this possible effect in the clinic, we retrospectively studied 182 high risk hematologic patients with invasive fungal infections (IFI) who were treated with L-AmB. Overall, 96 patients (52.7%) had possible, 52 (28.6%) probable and 34 (18.7%) proven IFI according to EORTC classification. Most had suspected or proven invasive aspergillosis. We compared patients with prior exposure to mold-active azoles (n=100) to those having not (n=82). The group with prior mold-active azoles included more patients with poor risk features for IFI as acute myeloid leukemia (p<0.05) and prolonged neutropenia (p<0.05). A favorable response in the IFI, defined as a complete or partial response, was achieved in 75% and 74.4% of patients in the whole cohort, and in 66% and 74.4% of patients with probable or proven IFI in the two groups. None of these differences were significant. Multivariate analysis showed that refractory baseline disease and renal dysfunction were adverse factors for response in the IFI (p<0.05). Survival was poorer for patients with prior broad spectrum azoles (p<0.05), and for those who did not recover from neutropenia (p<0.05). In conclusion, the effectiveness of treatment of breakthrough fungal infection with L-AmB is not likely to be affected by prior exposure to mold-active azoles prophylaxis, but survival largely depends on host and disease factors(AU)

Chemical composition and antioxidative activity of Echinophora platyloba DC. essential oil, and its interaction with natural antimicrobials against food-borne pathogens and spoilage organisms.

This study was undertaken to determine the chemical composition and antioxidative capacity of Echinophora platyloba DC. essential oil, and its antimicrobial potency against Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Salmonella typhimurium, Escherichia coli O157:H7, Pseudomonas aeruginosa, Candida albicans, Candida tropicalis, Rhodotorula rubra, and Rhodotorula mucilaginosa. The essential oil was analyzed by GC and GC-MS; and evaluated for its antioxidative and antimicrobial (singly or in combination with chitosan, nisin, monolaurin, or amphotericin B) activity. Thirty-three components were characterized representing 95.69% of the total oil composition in which thymol, trans-ocimene, carvacrol, and (E)-sesqui-lavandulol were the major constituents. The oil exhibited high scavenging (IC(50): 49.7 ± 2.3 µg/mL) and relative antioxidative activity (RAA%: 85.21 ± 0.4) in 1,1-diphenyl-2-picrylhydrazyl radicals and ß-carotene/linoleic acid bleaching assays, respectively. The oil showed antimicrobial activity against L. monocytogenes, B. cereus, B. subtilis, S. aureus, S. typhimurium, E. coli O157:H7, P. aeruginosa, C. albicans, C. tropicalis, R. Rubra, and R. mucilaginosa. Moreover, R. mucilaginosa and P. aeruginosa were the most susceptible and most resistant organisms, respectively. Regarding the checkerboard data, 47 fractional inhibitory concentration index (FICIs) (≤ 0.5) indicated synergistic, whereas 7 FICIs (>0.5 to 1) indicated additive effect. Consequently, E. platyloba DC. essential oil could be used as a recommended natural antioxidant and antimicrobial substance for food preservation.

Caracterización de aislamientos clínicos del complejo Cryptococcus neoformans-Cryptococcus gattii, del Estado Amazonas - Brasil / Characterization of clinical isolates of the Cryptococcus neoformans-Cryptococcus gattii species complex from the Amazonas State in Brazil

Antecedentes. La diferenciación y clasificación de las especies patógenas del género Cryptococcus aporta datos importantes para la asistencia clínica y para estudios epidemiológicos. Objetivos. El objetivo de este trabajo fue caracterizar 40 aislamientos clínicos del complejo Cryptococcus neoformans de pacientes que fueron atendidos en la Fundación de Medicina Tropical de Amazonas desde 2006 hasta 2008. Métodos. Se utilizaron métodos fenotípicos (producción de enzimas y pruebas de sensibilidad a los antifúngicos) y moleculares (URA5-RFLP). Resultados. Los pacientes con VIH/sida fueron los más afectados de criptococosis. Se observó que 31 (75,5%) y 9 (22,5%) de los aislamientos fueron Cryptococcus neoformans y Cryptococcus gattii, respectivamente. La producción de proteasa y fosfolipasa fue alta en la mayoría de las cepas. Utilizando la prueba de difusión en disco (CLSI M44-A) se observó que el 81, 35 y 100% de los aislamientos de C. neoformans fueron sensibles al fluconazol, itraconazol y amphotericin B, respectivamente, mientras que 78, 56 y 100% de los aislamientos de C. gattii fueron sensibles a estas sustancias. El valor promedio de la concentración mínima inhibitoria (CMI) para C. neoformans y C. gattii fue de 0,26 y 0,58mg/ml, respectivamente. Todos los aislamientos (9) de C. gattii presentaron un patrón de electroforesis compatible con el genotipo VGII, y todos los aislamientos (31) de C. neoformans presentaron el genotipo VNI. Conclusiones. Este estudio confirma la importancia del HIV/sida para la epidemiología de la criptococosis, la sensibilidad de los aislamientos a la anfotericina B y la alta prevalencia de los genotipos moleculares VNI y VGII en el norte de Brasil(AU)

Background. The differentiation and classification of pathogenic Cryptococcus species provides useful data for epidemiological studies and for the clinical diagnosis and treatment of patients. Aims. The aim of this study was to characterise 40 clinical Cryptococcus isolates obtained from patients at the Tropical Medicine Foundation of Amazonas (FMTAM) from 2006 to 2008. Methods. It was used phenotypic (i.e., enzyme production and antifungal resistance) and molecular biological (URA5-RFLP) experiments. Results. Patients with HIV/AIDS were most affected with cryptococcosis. Thirty-one (75.5%) of the clinical isolates were classified as Cryptococcus neoformans and 9 (22.5%) as Cryptococcus gattii. High amounts of protease and phospholipase enzymes were produced by most of the isolates. Using the disk diffusion test (CLSI M44-A), 81, 35 and 100% of the C. neoformans isolates were characterized as susceptible to fluconazole, itraconazole and amphotericin B, respectively, whereas 78, 56 and 100% of the C. gattii isolates were susceptible to these antimicrobial agents. The average of Minimal Inhibitory Concentration (MIC) for C. neoformans and C. gattii isolates was 0.26 and 0.58µg/mL, respectively. The 9 isolates of C. gattii had a fingerprint pattern comparable with the VGII molecular type, while all 31 isolates of C. neoformans presented with a pattern consistent with the VNI type. Conclusions. This study confirms the importance of HIV/AIDS for the cryptococcosis epidemiology, the susceptibility of the isolates to amphotericin B and the high prevalence of the molecular genotypes VNI and VGII in the north of Brazil(AU)

Development of respirable nanomicelle carriers for delivery of amphotericin B by jet nebulization.

The aim of the present work was to prepare and characterize chitosan-stearic acid conjugate nanomicelles for encapsulation of amphotericin B (AmB) and to evaluate the in vitro nebulization of the formulations. Water soluble chitosan was grafted to stearic acid (SA) chains via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide mediated coupling reaction. The chemical structure of depolymerized chitosan (DC)-SA copolymers and degree of amino substitution was determined by (1)H NMR. AmB was loaded in nanomicelles with a maximal encapsulation efficiency of 97%. The physicochemical properties and formation of polymeric micelles were studied by dynamic light scattering and fluorescence spectroscopy method. Nanomicelles possessed positive charges with mean particle sizes of 101-248 nm. AmB-loaded micelles were also characterized for their antifungal activity, aggregation state of the drug, nebulization efficiency and retention of AmB in the micelles after nebulization. The results indicated that encapsulation of AmB in DC-SA micelles could improve the antifungal activity of the drug in some of the cases. The nebulization efficiency was up to 56% and the fine particle fraction (FPF) varied from 40% to 52%. Since there was only a little change in encapsulation of the drug in micelles after nebulization, DC-SA micellar formulations can be a suitable choice for pulmonary delivery of AmB.