The in vitro susceptibilities against 11 antifungal drugs, including amphotericin B (Cayman Chemical, USA), anidulafungin (SelleckChem, USA), caspofungin (TargetMol, USA), micafungin (TargetMol), fluconazole (TargetMol), isavuconazole (TargetMol), itraconazole (TargetMol), posaconazole (Sigma-Aldrich), ravuconazole (Sigma-Aldrich), voriconazole (TargetMol) and flucytosine (Sigma-Aldrich), were determined by the microbroth dilution method according to the guidelines by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (test range: 0.0156–8 µg/mL for itraconazole and posaconazole; 0.0312–16 µg/mL for other drugs) [52 ] and our previous publication [53 (link)]. For echinocandins, minimum effective concentration (MEC) endpoints were recorded as the lowest drug concentrations in which abnormal, short and branched hyphal clusters were observed; whereas for the other antifungal agents, minimum inhibitory concentration (MIC) endpoints yielding no visible fungal growth by eyes were recorded. Strains Aspergillus flavus ATCC 204304 and CNM-CM-1813, Aspergillus fumigatus ATCC 204305, Candida albicans CNM-CL F8555, Candida parapsilosis ATCC 22019T as well as Pichia kudriavzevii CNM-CL-3403 and NRRL Y-413 were used as quality controls.
Posaconazole
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Japan, Spain, France, India, Belgium
Posaconazole is a laboratory product manufactured by Merck Group. It is an antifungal agent used in research and development applications.
Automatically generated - may contain errors
Lab products found in correlation
Itraconazole, by Merck Group (35 mentions)
Voriconazole, by Merck Group (33 mentions)
Amphotericin b, by Merck Group (27 mentions)
Fluconazole, by Merck Group (18 mentions)
Caspofungin, by Merck Group (16 mentions)
Ketoconazole, by Merck Group (11 mentions)
Isavuconazole, by Merck Group (10 mentions)
Anidulafungin, by Pfizer (7 mentions)
Micafungin, by Astellas Pharma (6 mentions)
Ravuconazole, by Merck Group (5 mentions)
Voriconazole, by Pfizer (5 mentions)
Micafungin, by Merck Group (4 mentions)
Clotrimazole, by Merck Group (4 mentions)
Tebuconazole, by Merck Group (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Benznidazole, by Merck Group (3 mentions)
Anidulafungin, by Merck Group (3 mentions)
Tween 20, by Merck Group (3 mentions)
Miconazole, by Merck Group (3 mentions)
Flucytosine, by Merck Group (2 mentions)
70 protocols using posaconazole
1
In Vitro Antifungal Susceptibility Assay
2
Antichagasic Drugs in Murine Chagas
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Benznidazole (Sigma, St Louis, MO) and Posaconazole (purified from oral suspension Noxafil) were solubilized in 10% solutol (Kolliphor, Sigma, St Louis, MO), and administered once a day orally, at 100 mg/kg for Benznidazole, and 20 mg/kg for Posaconazole. A solution of 10% solutol (just the vehicle) was administered to control, T. cruzi- infected groups. The treatment of mice acutely infected with T. cruzi Sylvio X10/7 strain started 6 days post-infection (dpi). For analysis at the chronic stage of infection, mice infected with T. cruzi Brazil strain were treated starting at 60 days post infection. Mice were treated for 21 days. In both approaches, survival, general health, and the weight of mice was monitored for one year after the end of treatment.
3
Synthesis and Formulation of Posaconazole
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Posaconazole was synthesized by Merck & Co. Inc., Kenilworth, NJ, USA; Posaconazole is a BCS Class II triazole medication to treat fungal infections [50 (link),51 (link)]. The polymer HPMCAS-M grade was a kind gift from ShinEtsu (Tokyo, Japan). The solvents methyl ethyl ketone (MEK), tetrahydrofuran (THF), acetone, methyl tert-butyl ether (MTBE), reagent alcohol, cyclohexane, and n-heptane were purchased from Fisher Scientific (Waltham, MA, USA).
4
Antifungal Susceptibility of C. africana Isolates
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All C. africana isolates obtained herein were tested for in vitro susceptibility to amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, and posaconazole (Sigma, St. Louis, MO, USA), caspofungin (Merck & Co., Inc.), and micafungin (Astellas Pharma) by using broth microdilution method as described in CLSI document M27-A3 and M27-S4. The MIC values were read following 24 h of incubation and determined visually as the lowest concentration in which prominent decrease in turbidity was observed excepting for amphotericin B which is defined as the lowest concentration in which the absence of turbidity is observed. The recently revised CLSI clinical breakpoints (CBPs) values for C. albicans were used as reference [28 (link)]. Quality control was performed as recommended in CLSI documents using C. parapsilosis ATCC22019 and C. krusei ATCC 6258.
5
Analytical Protocol for Posaconazole
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Posaconazole, 2-nitrophenyl octyl ether (NPOE), and α-cyano-4-hydroxycinnamic acid (CHCA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Trichloroacetic acid (TFA) was obtained from Alfa-Aesar (Ward Hill, MA, USA). Hydrophilic polyvinylidene difluoride (PVDF), nylon, polytetrafluoroethylene (PTFE), mixed cellulose ester (MCE), and hydrophobic polypropylene (PP) sheet membranes with a thickness of 200 μm and a pore size of 0.22 μm were purchased from Greattech Technology Ltd. (Taipei, Taiwan). All chemicals were used as received without further purification. Water was purified with a Millipore Synergy water purification system (Billerica, MA, USA) and used for all experiments.
A stock standard solution (1 mM) of Posaconazole was prepared in methanol and diluted to the desired concentrations with a 10 mM TFA aqueous solution (pH 2.2). The Posaconazole solutions were stored at −20 °C and protected from light for three months. CHCA solution (10 mg/mL) was freshly prepared in a 50% methanol/water solution containing 0.1% TFA.
A stock standard solution (1 mM) of Posaconazole was prepared in methanol and diluted to the desired concentrations with a 10 mM TFA aqueous solution (pH 2.2). The Posaconazole solutions were stored at −20 °C and protected from light for three months. CHCA solution (10 mg/mL) was freshly prepared in a 50% methanol/water solution containing 0.1% TFA.
6
Antifungal Susceptibility Testing Protocol
7
Antifungal Drugs Inhibit SARS-CoV-2 Infection
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The human type II alveolar epithelial cell line A549, the human epithelial carcinoma cell line A431, the interferon-deficient Vero cells and the Madin-Darby canine kidney (MDCK) cells type II were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% standardized fetal bovine serum (FBS Superior; Merck Cat. No. S0615), 2 mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. All cell lines were cultured in a humidified incubator at 37°C and 5% CO2. Itraconazole (Sigma) and posaconazole (Sigma) were solubilized in DMSO at 2 mg/ml. Prior to infection, cells were treated with 2 µg/ml Itraconazole or posaconazole for 16 h, or with 250 nM Bafilomycin A1 (Cayman-Cay-11038) for 2 h. For postinfection treatment, 2 µg/ml Itraconazole or posaconazole were added 2 h postinfection.
8
Antifungal Susceptibility Testing of CAPA
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To evaluate the susceptibility of various antifungal drugs, antifungal susceptibility testing of the CAPA isolates was performed using the EUCAST (European Committee for Antimicrobial Susceptibility Testing) reference microdilution method version 9.3.2 (54 ). Susceptibility to amphotericin B, isavuconazole, voriconazole, posaconazole, itraconazole, caspofungin, and micafungin was tested at 48 h. All antifungals were purchased from Sigma-Aldrich (Vienna, Austria). Candida parapsilosis ATCC 22019 and Aspergillus fumigatus ATCC 204305 were used as quality-control strains. In vitro tests were performed in duplicate. A categorization according to epidemiological cutoffs (ECOFFs) and clinical breakpoints (CBPs) was applied (24 (link)). Susceptible is <1 mg/L for amphotericin B, isavuconazole, and voriconazole; for the echinocandins, neither ECOFFS nor CBP are available. MICs falling within ± 2 dilutions (due to double testing) were converted to the highest concentration detected.
9
Invasive A. flavus Infection Models
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Four A flavus isolates were used in this study (Table 1 ). ATCC204304 was used for the primary development of new in vitro models of invasive A flavus infection. An additional 3 clinical A flavus isolates that had caused invasive disease were selected from the Special Mycology Laboratory (LEMI) (UNIFESP, Brazil). LEMI764 was isolated from a skin biopsy, LEMI1024 was isolated from a hepatic lesion, and LEMI1049 was isolated from nasal mucosa. Strains were identified as A flavus sensu stricto based on standard morphological and molecular studies, including sequence analysis of the internal transcribed spacer region, calmodulin, and β-tubulin genes [11 (link), 19 (link)].
Minimum inhibitory concentration (MIC) values were estimated according to European Committee on Antimicrobial Susceptibility Testing and Clinical Laboratory Standards Institute methodologies [20 , 21 ]. Compound with >98% purity was used for susceptibility testing. All susceptibility tests were performed as 10 independently conducted experiments for posaconazole (Sigma-Aldrich, Dorset, UK), voriconazole (Sigma-Aldrich, Dorset, UK), and F901318 (F2G Ltd., Manchester, UK).
Minimum inhibitory concentration (MIC) values were estimated according to European Committee on Antimicrobial Susceptibility Testing and Clinical Laboratory Standards Institute methodologies [20 , 21 ]. Compound with >98% purity was used for susceptibility testing. All susceptibility tests were performed as 10 independently conducted experiments for posaconazole (Sigma-Aldrich, Dorset, UK), voriconazole (Sigma-Aldrich, Dorset, UK), and F901318 (F2G Ltd., Manchester, UK).
10
CYP Inhibition by PC945 and Posaconazole
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PC945 and posaconazole (Sigma‐Aldrich, UK) were added to the pooled human liver microsomal samples (Pharmaron Rushden, UK) at concentrations of 0, 0.03, 0.1, 0.3, 1, 3, and 5 μmol/L. The mixtures were preincubated at 37°C for 0 or 30 minutes before the addition of selective CYP substrates. Microsomal reactions were terminated by adding 100 μL of cold (4°C) methanol containing appropriate internal standard. Aliquots (150 μL) of each sample were centrifuged and the supernatant was used for substrate analysis by ultra‐performance liquid chromatography with tandem mass spectrometry (UPLC‐MS/MS) on the day of incubations.
Change in substrate concentration was measured to determine the extent of CYP‐isoform inhibition. The 30‐minute preincubations were performed to determine whether there was time‐dependent inhibition of any CYP isoform (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4/5). Untreated control contained only solvent, dimethyl sulfoxide (≤0.5% [v/v]). Positive control contained a chemical inhibitor selective for each CYP enzyme in the presence of PC945 or posaconazole.
Change in substrate concentration was measured to determine the extent of CYP‐isoform inhibition. The 30‐minute preincubations were performed to determine whether there was time‐dependent inhibition of any CYP isoform (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4/5). Untreated control contained only solvent, dimethyl sulfoxide (≤0.5% [v/v]). Positive control contained a chemical inhibitor selective for each CYP enzyme in the presence of PC945 or posaconazole.
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