Effect of SDS on release of intracellular pneumocandin B0 in extractive batch fermentation of Glarea lozoyensis.

Pneumocandin B0 is a hydrophobic secondary metabolite that accumulates in the mycelia of Glarea lozoyensis and inhibits fungal 1,3-ß-glucan synthase. Extractive batch fermentation can promote the release of intracellular secondary metabolites into the fermentation broth and is often used in industry. The addition of extractants has been proven as an effective method to attain higher accumulation of hydrophobic secondary metabolites and circumvent troublesome solvent extraction. Various extractants exerted significant but different influences on the biomass and pneumocandin B0 yields. The maximum pneumocandin B0 yield (2528.67 mg/L) and highest extracellular pneumocandin B0 yield (580.33 mg/L) were achieved when 1.0 g/L SDS was added on the 13th day of extractive batch fermentation, corresponding to significant increases of 37.63 and 154% compared with the conventional batch fermentation, respectively. The mechanism behind this phenomenon is partly attributed to the release of intracellular pneumocandin B0 into the fermentation broth and the enhanced biosynthesis of pneumocandin B0 in the mycelia.

Functional characterization of host toxic EcdB transcription factor protein of echinocandin B biosynthetic gene cluster.

The ecdB is a transcription factor, located in the echinocandin B biosynthetic gene cluster of Emericella rugulosa NRRL11440. Here, we validated the ecdB mRNA sequence for functional expression and to explore the role of EcdB protein in the echinocandin B regulation. The sequence alignment study revealed that the ecdB coding sequence was found 75 bp shorter than the reference mRNA sequence. This coding sequence encodes for EcdB protein and comprises three conserved domains; DNA binding domain (DBD), coiled-coil domain, and signature middle homology region. The full-length and DBD (truncated) DNA sequences were expressed in Escherichia coli BL21(DE3) under different tested conditions. The expression of EcdB protein was found to be toxic, which curbs the cell growth. In contrast to truncated protein (GST:EcdB1-54), the full-length (GST:EcdB) protein was expressed at very low titer and not detectable in SDS-PAGE under the varying isopropyl ß-d-1-thiogalactopyranoside (IPTG), temperature, and media conditions. However, GST:EcdB1-54 was successfully purified under standard conditions (0.5 mM IPTG at 0.5OD) with 33 kDa expected size. The functionality of GST:EcdB1-54 was attained by electrophoretic mobility shift assay study as a clear band shifting showed with ecdA promoter. Taken together, we conclude that EcdB interacts with the ecdA promoter that reflected to require for echinocandin B regulation.

Development and validation of a liquid chromatography/tandem mass spectrometry method for determination of caspofungin in dried blood spots.

RATIONALE: A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for quantification of caspofungin in dried blood spots (DBS) was developed and validated. METHODS: The DBS samples were prepared by spotting whole blood onto Whatman 903 filter paper, drying at room temperature and extracting with 50% methanol and further cleaned by protein precipitation with acetonitrile. Roxithromycin was selected as internal standard, and the separation of the analytes with endogenous ingredients was accomplished on a Hypersil GOLD aQ column with a mobile phase composed of 0.1% formic acid (v/v) and methanol in gradient mode. The detection of the analytes was performed on a triple quadrupole mass spectrometer in positive electrospray ionization mode, and the following selective reaction monitoring (SRM) transitions were monitored: m/z 547.6 â†’ 538.7 and 837.4→ 679.4 for quantification of caspofungin and the internal standard, respectively. RESULTS: The total analytical time was 8 min for each run. The calibration curve exhibited a good linearity over the range from 0.2 to 20 µg/mL and the lower limit of quantification (LLOQ) was 0.2 µg/mL for caspofungin in DBS. The recoveries of caspofungin ranged from 62.64% to 76.69%, and no obvious matrix effect was observed. The intra- and inter-day precision and accuracy were within acceptable limits, and caspofungin in DBS was stable after storage at room temperature for 24 h and at -80°C for 30 days. There was no evident effect of the hematocrit value on the analysis of caspofungin. CONCLUSIONS: The proposed method presents an alternative to the conventional venous sampling method, and was successfully utilized for pharmacokinetics study of caspofungin in ICU patients.

Filter Adsorption of Anidulafungin to a Polysulfone-Based Hemofilter During CVVHD In Vitro.

Candidemia is frequent in critically ill patients, especially in combination with an acute kidney injury (AKI). Echinocandins generally are recommended for therapy of such infections. Recent studies found no need for dosage adjustment in patients with end-stage renal disease receiving hemodialysis, or patients with AKI receiving continuous venovenous hemofiltration. The aim of this in vitro study was to examine the adsorption of anidulafungin to the surface of the hemofilter during continuous venovenous hemodialysis (CVVHD) and its effect on anidulafungin concentrations. The concentration of anidulafungin in the dialyzed fluid, and the dialysate during CVVHD in vitro was examined using three different dialyzed fluids (saline; saline with 40 g/L human albumin; and a mixture of human erythrocytes and fresh frozen plasma). After the end of dialysis, the hemofilter was opened and portions of the filter capillaries were also analyzed to determine the amount of anidulafungin adsorbed. When dialyzing saline, about 99% of the anidulafungin used adsorbed to the hemofilter capillaries; in the experiments with saline with 40 g/L albumin, about 60% adsorbed to the hemofilter's surface, and when blood was dialyzed, 35% was found adsorbed after analyzing the filter capillaries. Anidulafungin was not detectable in the dialysate of any of the experiments, consequently the dialysis clearance was 0 mL/min. In conclusion, during CVVHD in vitro we found remarkable adsorption of anidulafungin to the hemofilter's surface, yet the effect on the tissue concentration needs further examination.

Cyclic colisporifungin and linear cavinafungins, antifungal lipopeptides isolated from Colispora cavincola.

Colisporifungin (1), a cyclic depsilipopeptide structurally related to the aselacins, and cavinafungins A and B, two linear peptides, were isolated from liquid culture broths of the hitherto unstudied fungus Colispora cavincola using a Candida albicans whole-cell assay as well as a bioassay to detect compounds potentiating the antifungal activity of caspofungin. The structural elucidation, including the absolute configuration of the new molecules, was accomplished using a combination of spectroscopic and chemical techniques, including 1D and 2D NMR, HRMS, and Marfey's analysis. The cyclic peptide colisporifungin displayed a strong potentiation of the growth inhibitory effect of caspofungin against Aspergillus fumigatus and, to a lesser extent, against Candida albicans. The linear peptides displayed broad-spectrum antifungal activities inhibiting growth of Candida species (MIC values 0.5-4 µg/mL) as well as A. fumigatus with a prominent inhibition of 8 µg/mL.

Preparative separation of echinocandin B from Aspergillus nidulans broth using macroporous resin adsorption chromatography.

Echinocandin B (ECB), an echinocandin type of lipopeptide antibiotic produced by Aspergillus nidulans, is a precursor for the synthesis of novel anti-fungal drug - anidulafungin. In this work, a separation strategy involving one-step macroporous resin adsorption chromatography was established for ECB purification from Aspergillus nidulans CCTCC M 2010275 fermentation broth. Among nine macroporous resin adsorbents tested, the non-polar resin HP-20 had the best adsorption and desorption performance. The static equilibrium adsorption data fitted well with the Langmuir equation, and the adsorption kinetic followed the pseudo-second order model. The separation parameters of ECB from broth were optimised by dynamic adsorption/desorption experiments with the column packed with HP-20 resin. Under optimal conditions, the purity increased by 3.8-fold from 23.2% in broth to 88.5% in eluent with 87.1% recovery yield by a one-step treatment. Our study provided a one-step and effective method for large-scale production of ECB, and offered references for separating other echinocandins from broth.

PROYECTO ÉPICO 2.0. Desarrollo de unas recomendaciones terapéuticas educacionales mediante metodología DELPHI en pacientes críticos adultos no neutropénicos con candidiasis invasiva en situaciones especiales / EPICO 2.0 PROJECT. Development of educational therapeutic recommendations using the DELPHI technique on invasive candidiasis in critically ill adult patients in special situations

Introducción. Aunque en la última década se ha mostrado una mejoría en el manejo de la candidiasis invasiva, todavía existe controversia, especialmente en el trtaamiento antifúngico en situaciones clínicas especiales. Objetivos. Identificar los principales conocimientos clínicos y elaborar recomendaciones con un alto nivel de consenso, necesarios para la elección del tratamiento antifúngico en situaciones especiales en sus diversos escenarios en pacientes adultos críticos no neutropénicos con candidiasis invasiva. Métodos. Cuestionario prospectivo español, que mide el consenso mediante la técnica Delphi, se realizó de forma anónima y por correo electrónico con 23 expertos multidisciplinarios nacionales, especialistas en infecciones fúngicas invasivas de cinco sociedades científicas nacionales, incluyendo Intensivistas, Anestesistas, Microbiólogos, Farmacólogos y Especialistas en Enfermedades Infecciosas que respondieron a 30 preguntas preparadas por el grupo de coordinación, tras una revisión exhaustiva de la literatura de los últimos cinco años. Los objetivos educativos contemplaron cuatro categorías, incluyendo candidiasis peritoneal, pacientes inmunodeprimidos, situaciones especiales y fracasos orgánicos. El nivel de acuerdo alcanzado entre los expertos en cada uno de las categorías debería superar el 75% para ser seleccionada. En un segundo término, después de extraer las recomendaciones de los temas seleccionados, se celebró una reunión presencial con más de 60 especialistas y se les solicitó la validación de las recomendaciones pre-seleccionadas. Mediciones y Resultados Principales. En un primer término, se realizó una pre-selección de 15 recomendaciones (Candidiasis peritoneal (3), Pacientes inmunosuprimidos (6), Situaciones especiales (3), Fracasos orgánicos (3)). Después de la segunda ronda, las siguientes 13 recomendaciones fueron validadas: Candidiasis peritoneal: Debido al mal pronóstico de la peritonitis candidiásica, se recomienda un adecuado control del foco infeccioso junto a un tratamiento antifúngico precoz y apropiado. Se recomienda iniciar un tratamiento antifúngico empírico en pacientes con peritonitis secundaria nosocomial y con factores de riesgo de colonización por Candida spp. o en aquellos pacientes con peritonitis terciaria. En la peritonitis candidiásica, se recomienda utilizar una equinocandina en los pacientes inestables o en aquellos que han recibido previamente azoles o en los que se aísla Candida spp. resistente a fluconazol. Pacientes inmunodeprimidos. En el tratamiento de la candidiasis invasora con azoles en un paciente con trasplante de órgano sólido, deben considerarse sus interacciones y hepatotoxicidad. En el paciente neutropénico, la duración del tratamiento de la candidemia debe ser de 14 días desde el primer cultivo negativo y hasta la normalización de la cifra de neutrófilos. En un paciente neutropénico inestable con candidemia y catéter venoso central de fácil recambio, es aconsejable la retirada del mismo. Situaciones especiales: En el tratamiento de la candidiasis invasiva en pacientes con disfunción hepática moderada (Child B) se recomienda utilizar equinocandinas (preferentemente anidulafungina o caspofungina con ajuste de dosis) y se debe evitar el uso de azoles. Aunque las interacciones farmacológicas de las equinocandinas son pocas, se recomienda revisar la medicación concomitante y en caso de posible interacción, utilizar preferentemente anidulafungina. Fracasos orgánicos: 1.-En lo que a seguridad se refiere las equinocandinas son la familia de antifúngicos de primera elección. Todas las equinocandinas son iguales para el tratamiento de los pacientes que necesitan técnicas continuas de depuración extrarrenal y no precisan ajuste de dosis. El uso de azoles precisa importantes ajustes de dosis en el paciente en tratamiento con técnica continua o intermitente de depuración extrarrenal. Conclusiones. El manejo de la candidiasis invasiva en pacientes de UCI requiere la aplicación de los conocimientos y destrezas que se detallan en nuestras recomendaciones. Estas recomendaciones ayudan a optimizar el tratamiento de los pacientes críticos con candidiasis invasiva en distintos escenarios y situaciones clínicas y mejorar su pronóstico, basados en la metodología DELPHI (AU)

Introduction. Although there has been an improved management of Invasive Candidiasis in the last decade, still controversial issues remain, especially in different therapeutic critical care scenarios. Objectives. We sought to identify the core clinical knowledge and to achieve high agreement recommendations required to care for critically ill adult patients with Invasive Candidiasis for antifungal treatment in special situations and different scenarios. Methods. Second Prospective Spanish survey reaching consensus by the Delphi technique, conducted anonymously by electronic e-mail in the first phase to 23 national multidisciplinary experts in invasive fungal infections from five national scientific societies including Intensivists, Anesthesiologists, Microbiologists, Pharmacologists and Infectious disease specialists, answering 30 questions prepared by a coordination group after a strict review of literature in the last five years. The educational objectives spanned four categories, including peritoneal candidiasis, immunocompromised patients, special situations and organ failures. The agreement among panellists in each item should be higher than 75% to be selected. In a second phase, after extracting recommendations from the selected items, a meeting was heldwith more than 60 specialists in a second round invited to validate the preselectedrecommendations. Measurements and Main Results. In the first phase, 15 recommendations were preselected (peritoneal candidiasis (3), immunocompromised patients (6), special situations (3) and organ failures (3)). After the second round the following 13 were validated: Peritoneal candidiasis (3): Source control and early adequate antifungal treatment is mandatory; empirical antifungal treatment is recommended in secondary nosocomial peritonitis with Candida spp colonization risk factors and in tertiary peritonitis. Immunocompromised patients (5): Consider hepatotoxicity and interactions before starting antifungal treatment with azoles in transplanted patients; treat candidemia in neutropenic adult patients with antifungal drugs at least 14 days after the first negative blood culture and until normalization of neutrophil count is achieved. Caspofungin, if needed, is the echinocandin with most scientific evidence to treat candidemia in neutropenic adult patients; Caspofungin is also the first choice drug to treat febrile candidemia; in neutropenic patients with candidemia remove catheter. Special situations (2): In moderate hepatocelular failure, patients with invasive candidiasis use echinocandins (preferably low doses of anidulafungin and caspofungin) and try to avoid azoles; in case of possible interactions review all of the drugs involved and preferably use Anidulafungin. Organ failures (3): Echinocandins are the safest antifungal drugs; reconsider the use of azoles in patients under renal replacement therapy; all of the echinocandins are accepted for the treatment of patients under continuous renal replacement therapy and do not require dosage adjustment. Conclusions. Treatment of Invasive Candidiasis in ICU patients requires a broad range of knowledge and skills as summarized in our recommendations. These recommendations may help to optimize the therapeutic management of these patients in special situations and different scenarios and improve their outcome based on the DELPHI methodology (AU)

A novel echinocandin MIG0310 with anticandida activity from newly isolated Fusarium sp. strain MS-R1.

AIM: To isolate and identify an anticandida compound from newly isolated Fusarium strain MS-R1 and characterization of its activity against standard and clinical strains of Candida. METHODS AND RESULTS: The fungal strain, Fusarium strain MS-R1, was isolated from soil. Molecular identification according to the internal transcribed spacer region of the rRNA gene sequence showed the strain to be strongly related to Fusarium brachygibbosum complex. Successive extractions of the active compound showed activity against Candida albicans, including clinical strains. Inhibition of a clinical strain of Candida tropicalis, but not Candida krusei and Candida glabrata, was also shown as tested by the broth microdilution assay. The compound was purified by liquid chromatography coupled with thin-layer chromatography and bioautography and characterized by nuclear magnetic resonance spectroscopy. It was confirmed to have the molecular formula C(48)H(66)O(18) and was identified as an echinocandin with a novel structure. CONCLUSION: A novel echinocandin-type antifungal metabolite, MIG0310, was isolated and characterized. This is a second echinocandin reported from a Fusarium species, indicating this genus to have wider range of echinocandin compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: The new compound may lead to new anticandidal drugs, broadening the treatment opportunities for Candida species, including those that are resistant to the current antifungal drugs.

Discovery and development of first in class antifungal caspofungin (CANCIDAS®)--a case study.

Covering: 1985 to 2001.This paper describes a fifteen year journey from concept to clinical discovery and development of the first in class caspofungin acetate (CANCIDAS®) a parenteral antifungal agent. Caspofungin is a semisynthetic derivative of pneumocandin B0, a naturally occurring, lipophilic cyclic peptide isolated from the fungus, Glarea lozoyensis. While the echinocandins had been previously studied for antifungal activity by several organizations, the class was dropped for a variety of reasons. Merck subsequently initiated a research program leading to the discovery and development of caspofungin. The multitude of challenges that ensued during the discovery and development process and which were successfully resolved by multi-disciplinary teams constitute the content of this article. The article consists of five sections that describe the discovery and development of caspofungin in chronological order: (i) discovery of the natural product pneumocandin B0 from fungal fermentations, (ii) fermentation development to improve the titer of pneumocandin B0 to make it commercially viable, (iii) semisynthetic modification by medicinal chemistry to successfully improve the properties of pneumocandin B0 leading to the discovery of caspofungin, (iv) development of commercial semisynthesis and purification and formulation development to improve stability and (v) clinical development and approval of CANCIDAS® as an antifungal drug which subsequently saved thousands of lives.

Development and validation of a stability-indicating high performance liquid chromatographic (HPLC) method for the determination of related substances of micafungin sodium in drug substances.

An isocratic, sensitive and stability-indicating high performance liquid chromatographic (HPLC) method for separation and determination of the related substances of micafungin sodium was developed. The chromatographic separation was achieved on Agilent Zorbax SB-C18 column (250 × 4.6 mm, 5 µm). Forced degradation study confirmed that the newly developed method was specific and selective to the degradation products. The performance of the method was validated according to the present ICH guidelines for specificity, linearity, accuracy, precision and robustness. Regression analysis showed correlation coefficient value greater than 0.999 for micafungin sodium and its six impurities. Limit of detection of impurities was in the range of 0.006%-0.013% indicating the high sensitivity of the newly developed method. Accuracy of the method was established based on the recovery obtained between 98.2% and 102.0% for all impurities. RSD obtained for the repeatability and intermediate precision experiments, was less than 1.0%. The method was successfully applied to quantify related substances of micafungin sodium in bulk drugs.

Chemical and physical modulation of antibiotic activity in emericella species.

The addition of epigenetic modifying agents and ion-exchange resins to culture media and solid-state fermentations have been promoted as ways to stimulate expression of latent biosynthetic gene clusters and to modulate secondary metabolite biosynthesis. We asked how combination of these treatments would affect a population of screening isolates and their patterns of antibiosis relative to fermentation controls. A set of 43 Emericella strains, representing 25 species and varieties, were grown on a nutrient-rich medium comprising glucose, casein hydrolysate, urea, and mineral salts. Each strain was grown in untreated agitated liquid medium, a medium treated with suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, 5-azacytidine, a DNA methylation inhibitor, an Amberlite non-ionic polyacrylate resin, and the same medium incorporated into an inert static vermiculite matrix. Species-inherent metabolic differences more strongly influenced patterns of antibiosis than medium treatments. The antibacterial siderophore, desferritriacetylfusigen, was detected in most species in liquid media, but not in the vermiculite medium. The predominant antifungal component detected was echinocandin B. Some species produced this antifungal regardless of treatment, although higher quantities were often produced in vermiculite. Several species are reported for the first time to produce echinocandin B. A new echinocandin analog, echinocandin E, was identified from E. quadrilineata.

An on-line solid phase extraction procedure for the routine quantification of caspofungin by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Extensive sets of data are required to investigate the potential use of a therapeutic drug monitoring with individualization of dosage of the antimycotic compound caspofungin. The goal was to develop an improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for this aim. METHODS: Following protein precipitation, on-line solid phase extraction was performed for sample preparation. As the internal standard compound the veterinary drug tylosin was used. A standard validation protocol was applied. RESULTS: Good reproducibility and accuracy of the method were observed. On-line solid phase extraction resulted in a convenient work-flow and good robustness of the method. CONCLUSIONS: This improved LC-MS/MS method was found reliable and convenient. It can be suggested for further work on the clinical pharmacology of caspofungin in the setting of clinical research laboratories.