Isolates detected as resistant by the screening method were further tested for antifungal susceptibility using the European Committee for Antimicrobial Susceptibility Testing (EUCAST) microdilution broth reference method [30 ]. All clinically available azole antifungal molecules were tested: itraconazole (ITZ) (Sigma-Aldrich); voriconazole (VCZ) (Sigma-Aldrich); posaconazole (PCZ) (MSD, Kenilworth, NJ, USA); and isavuconazole (ISA) (Basilea Pharmaceutica International Ltd., Basel, Switzerland). The number of conidia inoculated was 105 to 2.5 × 105 cfu/mL and the concentrations of each drug ranged from 0.016 to 8 mg/L. After 48 h of incubation at 37 °C, the minimum inhibitory concentration (MIC) was determined, both visually, as the lowest drug concentration that could completely inhibit fungal growth, and spectrophotometrically at 550 nm using a 90% growth inhibition endpoint. All tests were performed in triplicate. Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were included as quality controls.
Itraconazole itz
Manufactured by Merck Group
Sourced in United States
Itraconazole (ITZ) is a synthetic triazole antifungal agent. It is used as a reference standard in the analysis and quality control of pharmaceutical formulations containing itraconazole.
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Lab products found in correlation
Voriconazole vcz, by Merck Group (2 mentions)
Fluconazole, by Merck Group (2 mentions)
Amphotericin b amb, by Merck Group (2 mentions)
Voriconazole, by Merck Group (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Anidulafungin, by Pfizer (1 mentions)
Sodium deoxycholate, by Loba Chemie (1 mentions)
Sodium lauryl sulfate, by Loba Chemie (1 mentions)
Terbinafine trb, by Merck Group (1 mentions)
Milteforan, by Virbac (1 mentions)
Fluconazole flz, by Merck Group (1 mentions)
7 protocols using itraconazole itz
1
Antifungal Susceptibility Testing Protocol
2
Itraconazole Nanosuspension Formulation and Optimization
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Itraconazole (ITZ) was purchased from Sigma-Aldrich (Supplier: Wuhan Atomole Chemicals Co., Ltd., Wuhan, China) and jet-milled prior to use. Hydroxypropyl cellulose (HPC-SL; Nippon Soda Co., Ltd., Tokyo, Japan), polysorbate 80 (PS80; Kolb Distribution AG, Hedingen, Switzerland) and sodium dodecyl sulfate (SDS; BTC Europe GmbH, Monheim am Rhein, Germany) were used as stabilizers for the nanosuspensions. Milling beads (zirconium oxide, yttrium stabilized, density 6 g/mL, diameter 0.3–0.4 mm (except for investigation of the effect of bead size, see Section 3.2.3 )) were purchased from VMA Getzmann GmbH (Reichshof, Germany). For milling trials, 10 mL polypropylene (PP) vials from iphas (iphas Pharma-Verpackung GmbH, Würselen, Germany) were used.
3
Antifungal and Triterpene Stock Solutions
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Itraconazole (ITZ), Ketoconazole (KET), and Fluconazole (FLC) were purchased from Sigma Aldrich (Saint Louis, MO, USA). Stock solutions were prepared at a final concentration of 500 μg mL−1 in dimethyl sulfoxide (DMSO) 5% v/v. A stock solution of β-escin, 95% purity (Phytolab Vestenbergsgreuth, Germany), was prepared at a final concentration of 200 μg mL−1 in DMSO 5% v/v.
4
Antifungal Susceptibility Testing Protocol
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The CLSI broth microdilution (CLSI-BMD) method of M27-A3/S4 was used for antifungal susceptibility testing (AFST) (Clinical and Laboratory Standards Institute, M27-A3, 2008; Clinical and Laboratory Standards Institute, M27-S4, 2012). AFST included the following antifungal drugs, amphotericin B (AMB), fluconazole (FLZ), voriconazole (VRZ), itraconazole (ITZ) all from (Sigma-Aldrich, St. Louis, MO, U.S.A), micafungin (MFG) (Astellas, Munich, Germany) and anidulafungin (ANF) (Pfizer, NY, USA). Reference strains of C. parapsilosis (ATCC 22019) and C. krusei (ATCC 6258) were used for quality control purposes. Due to interlaboratory variation, caspofungin was not used in this study (Espinel-Ingroff et al., 2013 (link)). Plates containing antifungal drugs and isolates were incubated at 37°C for 24 h and data were recorded visually. MIC data were interpreted based on CLSI M60 (Clinical and Laboratory Standards Institute, M60, 2017). Isolates showing a minimum inhibitory concentration (MIC) ≥8 μg/ml were noted as resistant to FLZ, MFG, and ANF, while those with VRZ MIC ≥1 μg/ml were regarded resistant (Clinical and Laboratory Standards Institute, M60, 2017). AMB and ITZ MIC values were interpreted as epidemiological cut-off values (ECV), where non-wild-types (NWT) isolates had a MIC >2 and >0.5 μg/ml, respectively (Clinical and Laboratory Standards Institute, M60, 2017).
5
Itraconazole Formulation Development
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Itraconazole (ITZ) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Phospholipid 90 H was kindly donated by Lipoid GmbH (Ludwigshafen, Germany). Sodium deoxycholate (SDC) and sodium lauryl sulfate (SLS) were purchased from Loba Chemie (Mumbai, India). Hydroxyl propyl methyl cellulose (HPMC) was purchased from Alpha Chemica (Mumbai, India). All other chemicals were of analytical grade.
6
In vitro Antifungal Compound Assays
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For the in vitro assays, voriconazole (VCZ, Sigma-Aldrich), itraconazole (ITZ, Sigma-Aldrich), amphotericin B (AMB, Sigma-Aldrich), terbinafine (TRB, Sigma-Aldrich), and brilacidin (BRI, supplied by Innovation Pharmaceuticals) were diluted in dimethyl sulfoxide (DMOS); while miltefosine (MFS, Sigma-Aldrich), the milteforan active compound, was diluted in ethanol; and caspofungin (CSP, Sigma-Aldrich) was diluted in distilled water. milteforan (miltefosine 2%) was purchased from Virbac as an oral solution.
7
Antifungal Susceptibility Testing Protocol
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To determine the MIC values of each species, the broth microdilution method using CLSI-M27/A3 was followed [19 ]. AFST included the following antifungals; fluconazole (FLZ) (Sigma-Aldrich, St. Louis, MO, USA), voriconazole (VRZ) (Sigma-Aldrich, St. Louis, MO, USA), itraconazole (ITZ) (Sigma-Aldrich, St. Louis, MO, USA) anidulafungin (AND) (Pfizer, NY, USA), micafungin (MFG) (Astellas, Munich, Germany), and amphotericin B (AMB) (Sigma-Aldrich, St. Louis, MO, USA). MIC values were visually read after 24 h of incubation at 35 °C, and Pichia kudriavzveii (ATCC 6258) and C. parapsilosis (ATCC 22019) were used for quality control purposes. MIC data were interpreted in a species-specific manner as suggested [20 (link)].
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