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Background: Oral candidiasis is an opportunistic disease caused by fungi of the Candida genus. The occurrence of Candida spp. resistance to the commercial antifungal drugs points to the search for alternative treatments. Propolis has been successfully used in the treatment of infectious diseases for centuries. It has been proposed that an ultrasound pretreatment in the propolis extraction protocol can enhance the concentrations of molecules with antimicrobial activities in the final extract. Thus, this study aimed to compare the antifungal activity against oral Candida spp. isolates of green and red propolis extracts submitted or not to an ultrasound pretreatment before the extraction procedure., Methods: Candida spp. were isolated from denture stomatitis lesions and identified by sequencing. Oral Candida spp. isolates and reference strains were submitted to broth microdilution assays using commercial antifungals and Brazilian green and red propolis extracts submitted or not to an ultrasound pretreatment. Minimal Inhibitory Concentrations (MIC) and Minimal Fungicide Concentrations (MFC) were determined and biofilm formation interference was evaluated for resistant isolates., Results: C. albicans, Candida tropicalis and Candida dubliniensis were isolated from denture stomatitis lesions. Growth inhibition was observed in all Candida isolates incubated with all green and red propolis extracts. At lower doses, red propolis extracts presented significant antifungal activity. The ultrasound pretreatment did not promote an increase in the antifungal activity of green or red propolis. Three isolates, which were highly resistant to fluconazole and itraconazole, were susceptible to low doses of red propolis extracts. These same three specimens had their biofilm formation inhibted by red propolis ethanolic extract., Conclusions: Thus, red propolis can be faced as a promising natural product to be used in the auxiliary antifungal therapy of denture stomatitis., (© 2021. The Author(s).)

Background/aim: Itraconazole shows anticancer activity in various types of cancer but its underlying mechanism is unclear. We investigated the effect of itraconazole on membrane-associated lipids., Materials and Methods: To investigate the influences of itraconazole on cholesterol trafficking, cervical cancer CaSki cells were cultured with itraconazole and analyzed by Filipin staining followed by confocal microscopy. Effect on the glycerophospholipid profiles was analyzed by liquid chromatography/mass spectrometry (LC/MS)., Results: After itraconazole treatment, Filipin staining revealed cholesterol accumulation in the intracellular compartments, which was similar to the distribution after treatment of U18666A (cholesterol transport inhibitor). LC/MS analysis showed a significant decrease in phosphatidylserine levels and an increase in lysophosphatidylcholine levels in CaSki cells., Conclusion: Itraconazole inhibited cholesterol trafficking and altered the phospholipid composition. Alterations in the cell membrane can potentiate the anticancer activity of itraconazole., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Selumetinib (ARRY-142886), an oral, potent and highly selective allosteric mitogen-activated protein kinase kinase 1/2 inhibitor, is approved by the US Food and Drug Administration for the treatment of pediatric patients aged ≥2 years with neurofibromatosis type 1 with symptomatic, inoperable plexiform neurofibromas. A physiologically based pharmacokinetic (PBPK) model was constructed to predict plasma concentration-time profiles of selumetinib, and to evaluate the impact of coadministering moderate cytochrome P450 (CYP) 3A4/2C19 inhibitors/inducers. The model was also used to extrapolate pharmacokinetic exposures from older children with different body surface area to guide dosing in younger children. This model was built based on physiochemical data and clinical in vivo drug-drug interaction (DDI) studies with itraconazole and fluconazole, and verified against data from an in vivo rifampicin DDI study and an absolute bioavailability study. The pediatric model was updated by changing system-specific input parameters using the Simcyp pediatric module. The model captured the observed selumetinib pharmacokinetic profiles and the interactions with CYP inhibitors/inducers. The predictions from the PBPK model showed a DDI effect of 30% to 40% increase or decrease in selumetinib exposure when coadministered with moderate CYP inhibitors or inducers, respectively, which was used to inform dose management and adjustments. The pediatric PBPK model was applied to simulate exposures in specific body surface area brackets that matched those achieved with a 25 mg/m 2 dose in SPRINT clinical trials. The pediatric PBPK model was used to guide the dose for younger patients in a planned pediatric clinical study., (© 2021 The Authors. The Journal of Clinical Pharmacology published by Wiley Periodicals LLC on behalf of American College of Clinical Pharmacology.)

The resistance of Candida albicans to azole drugs represents a great global challenge. This study investigates the potential fungicidal effects of atorvastatin (ATO) combinations with fluconazole (FLU), itraconazole (ITR), ketoconazole (KET) and voriconazole (VOR) against thirty-four multidrug-resistant (MDR) C. albicans using checkerboard and time-kill methods. Results showed that 94.12% of these isolates were MDR to ≥ two azole drugs, whereas 5.88% of them were susceptible to azole drugs. The tested isolates exhibited high resistance rates to FLU (58.82%), ITR (52.94%), VOR (47.06%) and KET (35.29%), whereas only three representative (8.82%) isolates were resistant to all tested azoles. Remarkably, the inhibition zones of these isolates were increased at least twofold with the presence of ATO, which interacted in a synergistic (FIC index ≤ 0.5) manner with tested azoles. In silico docking study of ATO and the four azole drugs were performed against the Lanosterol 14-alpha demethylase enzyme (ERG11) of C. albicans. Results showed that the mechanism of action of ATO against C. albicans is similar to that of azole compounds, with a docking score (-4.901) lower than azole drugs (≥5.0) due to the formation a single H-bond with Asp 225 and a pi-pi interaction with Thr 229. Importantly, ATO combinations with ITR, VOR and KET achieved fungicidal effects (≥ 3 Log 10 cfu/ml reduction) against the representative isolates, whereas a fungistatic effect (≤ 3 Log 10 cfu/ml reduction) was observed with FLU combination. Thus, the combination of ATO with azole drugs could be promising options for treating C. albicans infection., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Itraconazole, an FDA-approved antifungal, has antitumor activity against a variety of cancers. We sought to determine the effects of itraconazole on esophageal cancer and elucidate its mechanism of action. Itraconazole inhibited cell proliferation and induced G 1 -phase cell-cycle arrest in esophageal squamous cell carcinoma and adenocarcinoma cell lines. Using an unbiased kinase array, we found that itraconazole downregulated protein kinase AKT phosphorylation in OE33 esophageal adenocarcinoma cells. Itraconazole also decreased phosphorylation of downstream ribosomal protein S6, transcriptional expression of the upstream receptor tyrosine kinase HER2, and phosphorylation of upstream PI3K in esophageal cancer cells. Lapatinib, a tyrosine kinase inhibitor that targets HER2, and siRNA-mediated knockdown of HER2 similarly suppressed cancer cell growth in vitro Itraconazole significantly inhibited growth of OE33-derived flank xenografts in mice with detectable levels of itraconazole and its primary metabolite, hydroxyitraconazole, in esophagi and tumors. HER2 total protein and phosphorylation of AKT and S6 proteins were decreased in xenografts from itraconazole-treated mice compared to xenografts from placebo-treated mice. In an early phase I clinical trial (NCT02749513) in patients with esophageal cancer, itraconazole decreased HER2 total protein expression and phosphorylation of AKT and S6 proteins in tumors. These data demonstrate that itraconazole has potent antitumor properties in esophageal cancer, partially through blockade of HER2/AKT signaling., (©2021 The Authors; Published by the American Association for Cancer Research.)

The retrograde signaling pathway is well conserved from yeast to humans, which regulates cell adaptation during stress conditions and prevents cell death. One of its components, RTG1 encoded Rtg1p in association with Rtg3p communicates between mitochondria, nucleus, and peroxisome during stress for adaptation, by regulation of transcription. The F-box motif protein encoded by YDR131C  constitutes a part of SCF Ydr131c -E3 ligase complex, with unknown function; however, it is known that retrograde signaling is modulated by the E3 ligase complex. This study reports epistasis interaction between YDR131C and RTG1, which regulates cell growth, response to genotoxic stress, decreased apoptosis, resistance to petite mutation, and cell wall integrity. The cells of ydr131cΔrtg1Δ genetic background exhibits growth rate improvement however, sensitivity to hydroxyurea, itraconazole antifungal agent and synthetic indoloquinazoline-based alkaloid (8-fluorotryptanthrin, RK64), which disrupts the cell wall integrity in Saccharomyces cerevisiae. The epistatic interaction between YDR131C and RTG1 indicates a link between protein degradation and retrograde signaling pathways., (© 2021 Wiley Periodicals LLC.)

Objective: To investigate the effect of arsenic disulfide (AS 2 S 2 ) combined with itraconazole on the proliferation, apoptosis and hedgehog pathway of diffuse large B-cell lymphoma (DLBCL) cells., Methods: The human DLBCL cell OCI-LY3 was treated with different concentrations of AS 2 S 2 and itraconazole. Cell proliferation inhibition was detected by CCK-8, cell apoptosis rate was determined by flow cytometry. The expression levels of BCL-2, BAX, SMO and GLi1 were detected by Western blot., Results: The DLBCL cell viability was decreased significantly at 24, 48 or 72 h as cultured with itraconazole. Along with the increasing of itraconazole concentration, the DLBCL cell viability was significantly reduced as compared with that in control group, and the results showed statistically significant(r=-0.690，r=-0.639, r=-0.833, r=-0.808, r=-0.578). The inhibitory and apoptosis rates of the cells were significantly increased as compared with those of the single drug-treated group after treated by the combination of itraconazole and AS 2 S 2 (P<0.05). The protein levels of SMO and Glil were significantly down-regulated after treated by arsenic disulfide and itraconazole alone(P<0.01). The protein expression levels of SMO and Glil was down-regulated in the combined-treatment group(P<0.01)., Conclusion: Itraconazole can inhibit proliferation of DLBCL cells in a concentration-and time-dependent manner. In addition, the combination of AS 2 S 2 and itraconazole show a synergistic effects, which may be related with the down-regulated protein expression of SMO and Glil of Hedgehog signaling pathway.

Mobocertinib (TAK-788) is an investigational oral tyrosine kinase inhibitor targeting epidermal growth factor receptor and human epidermal growth factor 2. A phase 1 open-label, 2-period, fixed-sequence, 2-part study (NCT03928327) characterized effects of a strong CYP3A4 inhibitor (itraconazole) and inducer (rifampin) on the pharmacokinetics (PK) of mobocertinib and its active metabolites, AP32960 and AP32914. Healthy volunteers (n = 12 per part) received a single dose of mobocertinib alone (20 mg, part 1; 160 mg, part 2) and with multiple doses of itraconazole 200 mg once daily (part 1) or rifampin 600 mg once daily (part 2). Coadministration of itraconazole with mobocertinib increased the combined molar area under the plasma concentration-time curve from time 0 to infinity (AUC 0-∞ ) of mobocertinib, AP32960, and AP32914 by 527% (geometric least-squares mean [LSM] ratio, 6.27; 90% confidence interval [CI], 5.20-7.56). Coadministration of rifampin with mobocertinib decreased the combined molar AUC 0-∞ of mobocertinib, AP32960, and AP32914 by 95% (geometric LSM ratio, 0.05; 90%CI, 0.04-0.07). Based on these results, the strong CYP3A inhibitor itraconazole and inducer rifampin significantly influenced the PK of mobocertinib and its active metabolites. Coadministration of mobocertinib with moderate and strong CYP3A inhibitors or inducers is not recommended in ongoing clinical trials., (© 2021 Takeda Pharmaceuticals International Co. Clinical Pharmacology in Drug Development published by Wiley Periodicals LLC on behalf of American College of Clinical Pharmacology.)

Background/aim: The anticancer mechanism of itraconazole remains unsolved; therefore, we studied itraconazole-induced alterations in specialized pro-resolving mediators (SPMs) in cancer cells., Materials and Methods: The human cervical squamous carcinoma cell line CaSki was cultured with or without 1 μM itraconazole. Liquid chromatography/mass spectrometry analysis was conducted to identify SPMs that were influenced by itraconazole. Cell growth experiments were conducted using itraconazole and inhibitors targeting the metabolic pathways of candidate SPMs., Results: Resolvin E3, resolvin E2, prostaglandin J2 (PGJ2), delta-12-PGJ2, and maresin 2 were identified as candidate SPMs. The 12/15-lipoxygenase inhibitor, which is involved in the conversion of 18-hydroxy-eicosapentaenoic acid to resolvin E3, attenuated the inhibitory effect of itraconazole. Inhibition of the PGJ2 metabolic pathway did not interfere with itraconazole treatment., Conclusion: The metabolic pathway of SPMs, including resolving E3, could be proposed as an anticancer target of itraconazole., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Recently, Cryptococcus and non- albicans Candida (NAC) have emerged as health-threatening pathogens for clinical fungal infections. Due to their increased resistance to existing antifungal drugs, novel antifungals are urgently needed. In this study, we evaluated the antifungal effect of VT-1161 and its comparators itraconazole and fluconazole against common fluconazole-sensitive or -resistant Cryptococcus and NAC strains. The tested strains were obtained from Chinese patients by the Invasive Fungal Infection Group within the past 2 years. The minimum inhibitory concentrations (MICs) of VT-1161 and other triazoles were measured according to the Clinical and Laboratory Standards Institute (CLSI) M27-Ed4 guidelines. We found that VT-1161 exhibited strong in vitro activity against Cryptococcus spp.. VT-1161 (geometric mean MIC = 0.024 μg/mL) was 21.7-fold and 104.5-fold more potent than itraconazole and fluconazole, respectively. Against the seven Cryptococcus neoformans isolates with higher fluconazole MICs (≥8 μg/mL based on the MIC 90 value of this azole), VT-1161 maintained potent activities, with MICs ranging between 0.031 and 0.5 μg/mL. For NAC spp., VT-1161 (geometric mean MIC = 0.099 μg/mL) was 6.0-fold and 11.4-fold more effective than itraconazole and fluconazole, respectively. There is a positive correlation of the MICs between VT-1161 and itraconazole/fluconazole. The MIC values of VT-1161 against Candida glabrata and Candida tropicalis were significantly lower than those of fluconazole, whereas for Candida parapsilosis the differences in the MIC values between VT-1161 and fluconazole were not statistically significant. The results showed that tetrazole VT-1161 might be a promising candidate for treating Cryptococcus and NAC infections.

We evaluated the in vitro susceptibility of Sporothrix schenckii s.str. and Sporothrix globosa to diphenyl diselenide (PhSe) 2 alone and in association with itraconazole (ITZ). Eight clinical isolates were tested in microdilution and checkerboard assays. (PhSe) 2 alone inhibited all isolates in concentration ≤ 8 µg/mL and was effective in killing one S. schenckii isolate. Inhibitory and fungicidal beneficial effects in its interaction with ITZ were shown against 87.5% (7/8) and 50% (4/8) of the isolates tested, respectively. Our study demonstrates the in vitro antifungal activity of (PhSe) 2 against two pathogenic Sporothrix species, suggesting studies of in vivo applications are warranted., (© 2021. Sociedade Brasileira de Microbiologia.)

Resistance to antibiotics because of misuse and overuse is one of the greatest public health challenges worldwide. Despite the introduction of advanced nanotechnology in the production of antibiotics, the choice of appropriate medicines is limited due to side effects such as blood coagulation, toxicity, low efficacy, and low biocompatibility; therefore, novel nanomaterial composites are required to counter these repercussions. We first introduce a facile method for synthesizing a h omobifunctional imidoester-coated n ano s pindle (HINS) zinc oxide composite for enhancement of antibiotic efficacy and reduction of toxicity and blood coagulation. The antibiotic efficacy of the composites is twice that of commercialized zinc nanoparticles; in addition, they have good biocompatibility, have increased surface charge and solubility owing to the covalent acylation groups of HI, and produce a large number of Zn + ions and defensive reactive oxygen species (ROS) that effectively kill bacteria and fungi. The synergistic effect of a combination therapy with the HINS composite and itraconazole shows more than 90% destruction of fungi in treatments with low dosage with no cytotoxicity or coagulation evident in intravenous administration in in vitro and in vivo experiments. Thus, HINS composites are useful in reducing the effect of misuse and overuse of antibiotics in the medical field.

For many fungal infections, in vitro susceptibility testing is used to predict if an isolate is resistant or susceptible to the antifungal agent used to treat the infection. For Madurella mycetomatis, the main causative agent of mycetoma, in vitro susceptibility testing currently is not performed on a routine basis. The current in vitro susceptibility testing method is labor-intensive, and sonication must be done to generate a hyphal inoculum. For endpoint visualization, expensive viability dyes are needed. Here, we investigated if the currently used in vitro susceptibility method could be adapted to make it amendable for use in a routine setting which can be used in low-income countries, where mycetoma is endemic. First, we developed a methodology in which hyphal fragments can be generated without the need for sonication, by comparing different bead beating methodologies. Next, in vitro susceptibility was assessed using standard broth microdilution assays as well as disc diffusion, Etest, and VIPcheck methodologies. We demonstrate that after a hyphal suspension is generated by glass bead beating, disc diffusion, Etest, and VIPcheck can be used to determine susceptibility of Madurella mycetomatis to itraconazole, posaconazole, and voriconazole. The MICs found with Etest were comparable to those obtained with our modified CLSI-based broth microdilution in vitro susceptibility assay for itraconazole and posaconazole. Furthermore, we found an inverse relationship between the zones of inhibition and MICs obtained with the Etest and those obtained by the modified CLSI broth microdilution technique.

Extracellular vesicles (EVs) are mediators of intercellular communication under both healthy and pathological conditions, including the induction of pro-metastatic traits, but it is not yet known how and where functional cargoes of EVs are delivered to their targets in host cell compartments. We have described that after endocytosis, EVs reach Rab7 + late endosomes and a fraction of these enter the nucleoplasmic reticulum and transport EV biomaterials to the host cell nucleoplasm. Their entry therein and docking to outer nuclear membrane occur through a tripartite complex formed by the proteins VAP-A, ORP3 and Rab7 (VOR complex). Here, we report that the antifungal compound itraconazole (ICZ), but not its main metabolite hydroxy-ICZ or ketoconazole, disrupts the binding of Rab7 to ORP3-VAP-A complexes, leading to inhibition of EV-mediated pro-metastatic morphological changes including cell migration behaviour of colon cancer cells. With novel, smaller chemical drugs, inhibition of the VOR complex was maintained, although the ICZ moieties responsible for antifungal activity and interference with intracellular cholesterol distribution were removed. Knowing that cancer cells hijack their microenvironment and that EVs derived from them determine the pre-metastatic niche, small-sized inhibitors of nuclear transfer of EV cargo into host cells could find cancer therapeutic applications, particularly in combination with direct targeting of cancer cells., Competing Interests: The United Kingdom patent applications GB1810556,9 and GB2014012.5 and United States provisional patent number US62/690,616 are pending. The authors declare no other competing interests., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Purpose: To develop a liquid crystal (LC) precursor that can be used as a novel vaginal delivery system for Itraconazole (ITZ) and evaluate its pharmacodynamics., Methods: The LC precursor was prepared by using phytantriol (PYT) as lipid matrix and N, N-dimethylformamide (DMAC) as solvent. Swelling studies were performed to assess the phase conversion ability. The formulations were characterized by crossed polarized light microscopy (CPLM), small-angle X-ray scattering (SAXS). Moreover, the rheological and in vitro drug release behavior were investigated. Then the vaginal retention time of ITZ in the optimal prescription was evaluated. Finally, the pharmacodynamics studies of the ITZ-loaded LC precursor were performed in a mouse model of vulvovaginal candidiasis (VVC)., Results: The LC precursor could transform to LC gels after administration into the vagina. Based on PLM and SAXS, the LC gels, formed after phase-conversion, were cubic LC. The LC precursor was non-Newtonian, while the LC gels exhibited a pseudo-plastic fluid behavior. In vitro release results revealed that F2 (68.0%) had a higher cumulative drug release than that of F1 (59.17%) at 72 h. Most of the LC gels could be retained in the vagina of mice for 24-36 h. Pharmacodynamics studies showed that there was only mild inflammation or no inflammatory stimulation in the control group. The ITZ-loaded LC precursor significantly improved the symptoms of vaginitis in mice and had a better therapeutic effect than that of the positive control group., Conclusions: The ITZ-loaded LC precursor would be a promising formulation for vaginal drug delivery.

Background: Dermatophytes showing reduced sensitivity to antifungal agents have emerged in several countries. One terbinafine resistant strain of Trichophyton rubrum, TIMM20092, also showed reduced sensitivity to itraconazole (ITC) and voriconazole (VRC). The expression of two genes (TruMDR2 and TruMDR3) encoding multidrug transporters of the ABC family was found to be highly up-regulated in this strain. Deletion of TruMDR3 in TIMM20092 abolished its resistance to VRC but only slightly reduced its resistance to ITC., Objectives: We examined the potential of T rubrum to develop resistance to ITC by analysing the mechanism of ITC resistance in TIMM20092., Methods: The deletion of TruMDR2 by gene replacement was performed in TIMM20092 and one TruMDR3-lacking mutant (∆TruMDR3) previously generated from TIMM20092. TruMDR2 single and TruMDR2/TruMDR3 double mutants (∆TruMDR2 and ∆TruMDR2/3) were successfully obtained, respectively., Results: The suppression of TruMDR2 was shown to abolish resistance to ITC in TIMM20092 and the TruMDR3-lacking mutant, strongly suggesting that TruMDR2 is a major contributor to ITC resistance in TIMM20092., Conclusions: Our study highlights the possible role of the ABC transporter TruMDR2 in ITC resistance of T. rubrum., (© 2021 Wiley-VCH GmbH.)

Ipatasertib is a pan-AKT inhibitor in development for the treatment of cancer. Ipatasertib was metabolized by CYP3A4 to its major metabolite, M1 (G-037720), and was a P-gp substrate and OATP1B1/1B3 inhibitor in vitro. A phase I drug-drug interaction (DDI) study ( n = 15) was conducted in healthy subjects to evaluate the effect of itraconazole (200-mg solution QD, 4 days), a strong CYP3A4 and P-gp inhibitor, on pharmacokinetics of ipatasertib (100-mg single dose). Itraconazole increased the C max and AUC 0 -∞ of ipatasertib by 2.3- and 5.5-fold, respectively, increased the half-life by 53%, and delayed the t max by 1 hour. The C max and AUC 0-72h of its metabolite M1 (G-037720) reduced by 91% and 68%, respectively. This study confirmed that CYP3A4 plays a major role in ipatasertib clearance. Furthermore, the interaction of ipatasertib with coproporphyrin (CP) I and CPIII, the two endogenous substrates of OATP1B1/1B3, was evaluated in this study. CPI and CPIII plasma levels were unchanged in the presence of ipatasertib, both at exposures of 100 mg and at higher exposures in combination with itraconazole. This indicated no in vivo inhibition of OATP1B1/1B3 by ipatasertib. Additionally, it was shown that CPI and CPIII were not P-gp substrates in vitro, and itraconazole had no effect on CPI and CPIII concentrations in vivo. The latter is an important finding because it will simplify interpretation of future DDI studies using CPI/CPIII as OATP1B1/1B3 biomarkers. SIGNIFICANCE STATEMENT: This drug-drug interaction study in healthy volunteers demonstrated that CYP3A4 plays a major role in ipatasertib clearance, and that ipatasertib is not an organic anion transporting polypeptide 1B1/1B3 inhibitor. Furthermore, it was demonstrated that itraconazole, an inhibitor of CYP3A4 and several transporters, did not affect CPI/CPIII levels in vivo. This increases the understanding and application of these endogenous substrates as well as itraconazole in complex drug interaction studies., (Copyright © 2021 by The Author(s).)

Chromoblastomycosis (CBM) is a chronic subcutaneous infection caused by genera of melanized fungi: Fonsecaea , Cladophialophora , Phialophora , Exophiala , and Rhinocladiella . Melanin is a virulence factor known to influence antifungal susceptibility. A specific inhibitor of melanin biosynthesis is tricyclazole. The aim of this study was to evaluate the effect of melanin inhibition on antifungal susceptibility of chromoblastomycosis agents and describe the susceptibility profiles of some unusual CBM agents. Seventy-six clinical isolates, representing 13 species of the five main genera of CBM agents, were studied. The antifungal susceptibility testing was performed according to the M38-A2 protocol of CLSI ( Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi , 3rd ed., CLSI Standard M38 , 2017). In the melanin inhibition test, 16 mg/liter of tricyclazole was added to the medium used in the inoculum preparation and the susceptibility assay. CBM agents were less susceptible to amphotericin B than azoles and terbinafine. The unusual species showed similar susceptibility profiles to those of other species of the same genera. With tricyclazole exposure, MICs of terbinafine, posaconazole, and itraconazole for Fonsecaea spp. significantly decreased ( P < 0.05). For Phialophora spp., this reduction was significant for posaconazole and itraconazole. For the other genera, there was a reduction in MICs of terbinafine and itraconazole; however, the statistical tests were not significant. Melanin inhibition can increase the antifungal susceptibility of most CBM agents to itraconazole and terbinafine, the main drugs used in the disease treatment. This increased susceptibility may open up new possibilities for therapy in refractory cases of CBM and/or cases caused by resistant fungal strains. Further studies are needed to confirm the same results in vivo .

We isolated ascomycetous yeasts including Candida species, that originally belonged to the genus Candida, from bulk milk in the Aichi area of Japan, and determined the minimum inhibitory concentrations (MICs) of antifungal drugs on these isolates by conducting E-tests. We isolated 7 human pathogenic species (14 isolates) from 14 bulk milk samples: 5 Candida species of yeasts, and 2 Candida-related species. Two isolates of C. albicans and C. inconspicua were resistant to fluconazole (MIC >32 mg/l). One isolate of C. krusei was resistant to both azoles (fluconazole: >256 mg/ml and itraconazole: 4 mg/l). One isolate of C. catenulata might be resistant to amphotericin B (>32 mg/l).

Anisotropic microparticles containing flower like morphologies have recently attracted significant attention due to their potentially varied application range. Aim of the present work was to build an anisotropic drug delivery system (ASLMMSs) for the encapsulation and enhancement of antifungal activity of a hydrophobic antifungal drug i.e. itraconazole (IRL) with the combination of lipophilic (lipids) as well as hydrophilic (mannitol) materials. Encapsulation efficiency (EE) due to glyceryl monosteatrate (G.ASLMMSs) was 89.02%, whereas Precirol ATO5 formulation (P.ASLMMSs) showed 96.98% EE. FTIR analysis discovered the hydrogen bonding and Vander Waal's forces between lipids and mannitol, while evaluation of XRD data revealed the detailed structural and microstructural characterization indicating the crystallinity of final formulations. Observation under SEM revealed that the final formulations grew in the form of flower like morphologies. These flower-like structures were more obvious for P.ASLMMSs. The increment in dissolution rate (>80% in 6h) could be attributed to the mannitol. Dilutions of Itraconazole loaded anisotropic solid lipid mannitol microstructures (ASLMMSs) in water at concentration range of (500μg/mL, 250μg/mL, 125μg/mL and 75μg/mL) exhibited increased antifungal activity, while free IRL dilution in water showed no zone of inhibition. Both formulations, specifically P.ASLMMSs could signify a promising drug delivery system for lipophilic antifungal drugs.

Although vaccination campaigns are currently being rolled out to prevent coronavirus disease (COVID-19), antivirals will remain an important adjunct to vaccination. Antivirals against coronaviruses do not exist, hence global drug repurposing efforts have been carried out to identify agents that may provide clinical benefit to patients with COVID-19. Itraconazole, an antifungal agent, has been reported to have activity against animal coronaviruses. Using cell-based phenotypic assays, the in vitro antiviral activity of itraconazole and 17-OH itraconazole was assessed against clinical isolates from a German and Belgian patient infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Itraconazole demonstrated antiviral activity in human Caco-2 cells (EC 50  = 2.3 µM; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay). Similarly, its primary metabolite, 17-OH itraconazole, showed inhibition of SARS-CoV-2 activity (EC 50  = 3.6 µM). Remdesivir inhibited viral replication with an EC 50  = 0.4 µM. Itraconazole and 17-OH itraconazole resulted in a viral yield reduction in vitro of approximately 2-log 10 and approximately 1-log 10 , as measured in both Caco-2 cells and VeroE6-eGFP cells, respectively. The viral yield reduction brought about by remdesivir or GS-441524 (parent nucleoside of the antiviral prodrug remdesivir; positive control) was more pronounced, with an approximately 3-log 10 drop and >4-log 10 drop in Caco-2 cells and VeroE6-eGFP cells, respectively. Itraconazole and 17-OH itraconazole exert in vitro low micromolar activity against SARS-CoV-2. Despite the in vitro antiviral activity, itraconazole did not result in a beneficial effect in hospitalized COVID-19 patients in a clinical study (EudraCT Number: 2020-001243-15)., (© 2021 Wiley Periodicals LLC.)

Purpose: Pyrotinib, an irreversible human epidermal growth factor receptor 2 (HER2), is a epidermal growth factor receptor double-target tyrosine kinase inhibitor used for treating HER2-positive breast cancer. This study aimed to evaluate the impact of the strong CYP3A4 inhibitor itraconazole on the safety and pharmacokinetics of pyrotinib in Chinese healthy adults., Patients and Methods: This was an open-label, randomized, self-control study. Eighteen healthy adults were included in this trial. They received a single 80 mg dose of pyrotinib orally on days 1 and 9, and a 200 mg once-daily dose of itraconazole on days 6 through 22. Blood samples were obtained, and the drug concentration was detected using liquid chromatography/tandem mass spectrometry., Results: Compared with pyrotinib alone, the exposure to pyrotinib co-administered with itraconazole substantially increased, and the C max and AUC 0-t increased by 2.78- and 10.8-fold, respectively. No serious adverse events were reported in this trial, and no participant dropped out of the trial because of adverse events., Conclusion: The exposure to pyrotinib was substantially affected by the action of itraconazole. The concomitant use of pyrotinib with itraconazole might require dose modification of pyrotinib. All treatments were well tolerated in healthy participants., Clinical Trial Registry: http://www.chinadrugtrials.org.cn/clinicaltrials.prosearch.dhtml, CTR20191866., Competing Interests: The authors are accountable for the content and writing of this manuscript and report no conflicts of interest related to this study., (© 2021 Liu et al.)

Background and Purpose: The SARS-COV-2 pandemic and the global spread of coronavirus disease 2019 (COVID-19) urgently call for efficient and safe antiviral treatment strategies. A straightforward approach to speed up drug development at lower costs is drug repurposing. Here, we investigated the therapeutic potential of targeting the interface of SARS CoV-2 with the host via repurposing of clinically licensed drugs and evaluated their use in combinatory treatments with virus- and host-directed drugs in vitro., Experimental Approach: We tested the antiviral potential of the antifungal itraconazole and the antidepressant fluoxetine on the production of infectious SARS-CoV-2 particles in the polarized Calu-3 cell culture model and evaluated the added benefit of a combinatory use of these host-directed drugs with the direct acting antiviral remdesivir, an inhibitor of viral RNA polymerase., Key Results: Drug treatments were well-tolerated and potently impaired viral replication. Importantly, both itraconazole-remdesivir and fluoxetine-remdesivir combinations inhibited the production of infectious SARS-CoV-2 particles > 90% and displayed synergistic effects, as determined in commonly used reference models for drug interaction., Conclusion and Implications: Itraconazole-remdesivir and fluoxetine-remdesivir combinations are promising starting points for therapeutic options to control SARS-CoV-2 infection and severe progression of COVID-19., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Candida albicans is one of the most common clinical pathogenic microorganisms and it is becoming a serious health threat, particularly to immunocompromised populations. Drug resistance of Candida species has also frequently emerged, and combination therapy for fungal infections has attracted considerable attention. In this study, we established the Qinling Mountains myxobacterial secondary metabolites library and a synergic assay in combination with ketoconazole against C. albicans was introduced for metabolites screening. Two active compounds with synergic anticandidal activities were obtained, which were identified as trans-resveratrol and cis-resveratrol. According to our study, resveratrol can reduce the dosage to 1/64 of ketoconazole as well as itraconazole. Furthermore, synergistic anticandidal activity of resveratrol combined with azoles was verified against a panel of clinical C. albicans isolates, and the combination strategy enhanced the azoles susceptibility of three fluconazole-resistant isolates. These findings suggest that resveratrol enhances the efficacy of azoles and provides a promising application in therapy of C. albicans infection., (© 2021 The Society for Applied Microbiology.)

Filamentous fungus Purpureocillium lilacinum is an emerging pathogen that infects immunocompromised and immunocompetent individuals and is resistant to several azole molecules. Although azole resistance mechanisms are well studied in Aspergillus sp. and Candida sp., there are no studies to date reporting P. lilacinum molecular response to these molecules. The aim of this study was to describe P. lilacinum molecular mechanisms involved in antifungal response against fluconazole and itraconazole. Transcriptomic analyses showed that gene expression modulation takes place when P. lilacinum is challenged for 12 h with fluconazole (64 μg/mL) or itraconazole (16 μg/mL). The antifungals acted on the ergosterol biosynthesis pathway, and two homologous genes coding for cytochrome P450 51 enzymes were upregulated. Genes coding for efflux pumps, such as the major facilitator superfamily transporter, also displayed increased expression in the treated samples. We propose that P. lilacinum develops antifungal responses by raising the expression levels of cytochrome P450 enzymes and efflux pumps. Such modulation could confer P. lilacinum high levels of target enzymes and could lead to the constant withdrawal of antifungals, which would force an increase in the administration of antifungal medications to achieve fungal morbidity or mortality. The findings in this work could aid in the decision-making for treatment strategies in cases of P. lilacinum infection.

Nadolol is a hydrophilic and nonselective β-adrenoceptor blocker with a bioavailability of 30%, relatively longer half-life, negligible metabolism, and predominant renal excretion. Previous studies have reported that nadolol is a substrate of P-glycoprotein, and the coadministration with itraconazole, a typical P-glycoprotein inhibitor, results in elevated plasma concentrations and cumulative urinary excretion of nadolol. In this study, we assessed whether measurements of urinary-excreted nadolol can be an alternative method of plasma pharmacokinetics for P-glycoprotein-mediated drug interactions in humans. We reanalyzed the pooled data set of plasma concentration and urinary excretion of nadolol from our previous clinical studies in a total of 32 healthy Japanese adults. The area under the plasma concentration-time curve from 0 to infinity (AUC 0-∞ ) of nadolol in individual subjects was significantly correlated with the maximum plasma concentration (r = 0.80, P < .01) and the cumulative amount excreted into urine (A e ) at 4 (r = 0.51, P = .01), 8 (r = 0.63, P < .01), 24 (r = 0.75, P < .01), and 48 (r = 0.77, P < .01) hours. Significant correlations were also observed between the AUC and A e during the same respective periods. In the drug interactions of nadolol with itraconazole, rifampicin, a well-known P-glycoprotein inducer, or grapefruit juice, there were significant correlations between the differences in AUC 0-48 and those in A e, 0-48 from the controls in individual subjects. These results suggest that the measurements of urinary excretion of nadolol can be employed as a sensitive and reliable alternative to plasma pharmacokinetics for the evaluation of P-glycoprotein-mediated drug interactions., (© 2021, The American College of Clinical Pharmacology.)

Objective: The aim of the study was to investigate whether inhibition of Sonic Hedgehog (SHH) pathway would prevent progression of Barrett's Esophagus (BE) to esophageal adenocarcinoma., Background: The hedgehog signaling pathway is a leading candidate as a molecular mediator of BE and esophageal adenocarcinoma (EAC). Repurposed use of existing off-patent, safe and tolerable drugs that can inhibit hedgehog, such as itraconazole, could prevent progression of BE to EAC., Methods: The efficacy of itraconazole was investigated using a surgical rat reflux model of Barrett's Metaplasia (BM). Weekly intraperitoneal injections of saline (control group) or itraconazole (treatment group; 200 mg/kg) were started at 24 weeks postsurgery. Esophageal tissue was harvested at 40 weeks. The role of the Hh pathway was also evaluated clinically. Esophageal tissue was harvested after 40 weeks for pathological examination and evaluation of the SHH pathway by immunohistochemistry., Results: BM was present in control animals 29 of 31 (93%) versus itraconazole 22 of 24 (91%). EAC was significantly lower in itraconazole 2 of 24 (8%) versus control 10 of 31 (32%), respectively (P = 0.033). Esophageal SHH levels were lower in itraconazole vs control (P = 0.12). In esophageal tissue from humans with recurrent or persistent dysplastic BE within 24 months of ablative treatment, strong SHH and Indian Hedgehog expression occurred in distal BE versus proximal squamous epithelium, odds ratio = 6.1 (95% confidence interval: 1.6, 23.4) and odds ratio = 6.4 (95% confidence interval: 1.2, 32.8), respectively., Conclusion: Itraconazole significantly decreases EAC development and SHH expression in a preclinical animal model of BM. In humans, BE tissue expresses higher SHH, Indian Hedgehog, and bone morphogenic protein levels than normal squamous esophageal epithelium., Competing Interests: The authors report no conflicts of interest., (Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Vulvovaginal candidiasis caused by Candida species is a prevalent fungal infection among women. It is believed that the pathogenesis of Candida species is linked with the production of biofilm which is considered a virulence factor for this organism. The aim of this study was molecular identification, antifungal susceptibility, biomass quantification of biofilm, and detection of virulence markers of Candida species., Methods: We investigated the molecular identification of 70 vaginal isolates of Candida species, antifungal resistance to amphotericin B, fluconazole, itraconazole, and voriconazole according to CLSI M27-A3 and M27-S4, biofilm formation, and frequency analysis of biofilm-related ALS1, ALS3, and HWP1 genes., Results: Our findings showed that the most common yeast isolated from vaginal discharge was C . albicans (67%), followed by the non- Candida albicans species (33%). All C . albicans complex isolates were confirmed as C . albicans by HWP-PCR, and all isolates of the C . glabrata complex were revealed to be C . glabrata sensu stricto using the multiplex PCR method. FLC resistance was observed in 23.4% of C . albicans and 7.7% of C . glabrata . The resistance rate to ITC was found in 10.6% of C . albicans . The frequency of ALS1, ALS3, and HWP1 genes among Candida species was 67.1%, 80%, and 81.4%, respectively. Biofilm formation was observed in 54.3% of Candida species, and the highest frequency detected as a virulence factor was for the ALS3 gene (97.3%) in biofilm-forming species. Discussion . Our results showed the importance of molecular epidemiology studies, investigating antifungal susceptibility profiles, and understanding the role of biofilm-related virulence markers in the pathogenesis of Candida strains., Competing Interests: The authors reported that they have no conflicts of interest in this paper., (Copyright © 2021 Faezeh Mohammadi et al.)

Aspergillus niger is an opportunistic pathogen commonly found in a variety of indoor and outdoor environments. An environmental isolate of A. niger from a pig farm was resistant to itraconazole, and in-depth investigations were conducted to better understand cellular responses that occur during growth when this pathogen is exposed to an antifungal. Using a combination of cultivation techniques, antibiotic stress testing, and label-free proteomics, this study investigated the physiological and metabolic responses of A. niger to sublethal levels of antifungal stress. Challenging A. niger with itraconazole inhibited growth, and the MIC was estimated to be > 16 mg · liter -1 Through the proteome analysis, 1,305 unique proteins were identified. During growth with 2 and 8 mg · liter -1 itraconazole, a total of 91 and 50 proteins, respectively, were significantly differentially expressed. When challenged with itraconazole, A. niger exhibited decreased expression of peroxidative enzymes, increased expression of an ATP-binding cassette (ABC) transporter most likely involved as an azole efflux pump, and inhibited ergosterol synthesis; however, several ergosterol biosynthesis proteins increased in abundance. Furthermore, reduced expression of proteins involved in the production of ATP and reducing power from both the tricarboxylic acid (TCA) and glyoxylate cycles was observed. The mode of action of triazoles in A. niger therefore appears more complex than previously anticipated, and these observations may help highlight future targets for antifungal treatment., (Copyright © 2021 American Society for Microbiology.)

Faldaprevir (FDV), a substrate of CYP3A/P-glycoprotein (P-gp), is a selective inhibitor of the hepatitis C virus (HCV) NS3/4 protease. FDV is currently under clinical development for application in interferon-free treatment regimens for patients with chronic HCV infection. Understanding the drug-drug interaction potential of FDV is critical, as certain drug combinations may facilitate the more rapid achievement of steady-state-that is, the ideal drug concentration and balanced metabolic cycle of absorption and elimination that optimize drug efficacy. We thus conducted this study to investigate the effect of itraconazole (ICZ), a strong inhibitor of CYP3A and a moderate inhibitor of P-gp, on the pharmacokinetics (PK) of FDV. Eighteen healthy male and female volunteers participated in this open-label, fixed-sequence study. FDV 120 mg twice daily (BID) was administered on Day 1, followed by 120 mg once daily (QD) from Day 2 until the end of the 10-day study; after 6 days of FDV alone, ICZ 200 mg was added to FDV for an additional 4 days (BID on Day 7 and QD from Day 8 to Day 10). Intensive PK sampling was performed after 6 days of FDV treatment and again after 4 days of combined FDV/ICZ treatment. The adjusted geometric mean (gMean) ratios (%) of area under the concentration curve over dosing interval at steady-state (AUC τ, ss ) and maximal concentration at steady-state (C max, ss ) for combined FDV/ICZ treatment vs. FDV treatment alone were 198.6% and 180.6%, respectively, with 90% confidence intervals (CIs) of 182.4-216.1 and 165.7-196.9. Administration of FDV alone or in combination with ICZ was observed to be safe and well-tolerated. Co-administration with ICZ, however, resulted in an approximately two-fold increase in FDV steady-state exposure. Furthermore, FDV required no dosage adjustment when co-administered with ICZ.

Cryptococcus species is an opportunistic yeast pathogen and classified into different molecular types according to typing techniques including multilocus sequence typing (MLST). The study aimed to investigate the genotypes of environmental Cryptococcus isolates using MLST and the relationship between the in vitro antifungal susceptibility and sequence types of isolates. Genotyping Cryptococcus isolates was performed by the MLST method at seven nuclear loci. Antifungal susceptibility was determined by using CLSI broth micro-dilution method for amphotericin B, fluconazole, itraconazole, voriconazole, flucytosine, and luliconazole. Seven sequence types (ST) were detected using MLST analysis, with the most frequent (50%) ST77, followed by ST4 (16.7%) among 30 C. neoformans isolates. All antifungals demonstrated excellent activity against isolates, except for itraconazole and amphotericin B that were non-wild type against 53.3% and 10% of isolates, respectively. Although seven sequence types belonging to C. neoformans isolates were detected, ST77 was the main sequence type in Ahvaz. Also, non-wild type isolates were only found against itraconazole and amphotericin B.

The poor solubility of itraconazole (ITZ) has limited its efficacy in the treatment of vaginal candidiasis. Accordingly, the improvement of ITZ solubility using a solid dispersion technique was important to enhance its antifungal activity. Besides, as the purpose of this research was to develop local-targeting formulations, bioadhesive-thermosensitive in situ vaginal gel combined with the gel-flake system was found to be the most suitable choice. To obtain optimum solubility, entrapment efficiency, and drug-loading capacity, optimization of solid dispersion (SD) and gel-flake formulations of ITZ was performed using a composite central design. The results showed that the optimized formulation of SD-ITZ was able to significantly enhance its solubility in both water and simulated vaginal fluid to reach the values of 4.211 ± 0.23 and 4.291 ± 0.21 mg/mL, respectively. Additionally, the optimized formulation of SD-ITZ gel flakes possessed desirable entrapment efficiency and drug-loading capacity. The in situ vaginal gel containing SD-ITZ gel flakes was prepared using PF-127 and PF-68, as the gelling agents, with the addition of hydroxypropyl methylcellulose (HPMC) as the mucoadhesive polymer. It was found that the obtained in situ vaginal gel provided desirable physicochemical properties and was able to retain an amount of more than 4 mg of ITZ in the vaginal tissue after 8 h. Importantly, according to the in vivo antifungal activity using infection animal models, the incorporation of the solid dispersion technique and gel-flake system in the formulation of the bioadhesive-thermosensitive in situ vaginal gel led to the most significant decrease of the growth of Candida albicans reaching <1 log colony-forming units (CFU)/mL or equivalent to <10% of the total colony after 14 days, indicating the improvement of ITZ antifungal activity compared to other treated groups. Therefore, these studies confirmed a great potential to enhance the efficacy of ITZ in treating vaginal candidiasis. Following these findings, several further experiments need to be performed to ensure acceptability and usability before the research reaches the clinical stage.

Sporotrichosis, the most common subcutaneous mycosis in several countries, is caused by the dimorphic fungus, Sporothrix spp. Given some limitations in the treatment of this disease, and the high potential of nikkomycin Z (NikZ) as an antifungal against dimorphic fungi, this study aimed to evaluate the in vitro susceptibility of Sporothrix spp. to NikZ alone and with the drug of choice, itraconazole (ITZ). Seventeen clinical isolates of three Sporothrix spp. species (10 S. brasiliensis, six S. schenckii sensu stricto and one S. globosa) were tested in microdilution and checkerboard assays. Minimal inhibitory concentration (MIC), minimal fungicidal concentration (MFC), fractional inhibitory and fungicidal concentration indexes (FICi and FFCi) were analyzed. MIC of NikZ alone could be determined against S. globosa (12.5 μg/ml) and against 67% (4/6) and 30% (3/10) of the S. schenckii sensu stricto and S. brasiliensis isolates, respectively (≤ 400 μg/ml). Synergism with ITZ was showed against almost all the isolates tested (94%; 16/17), including reversing resistance to ITZ alone in some isolates. Our study shows the potential of NikZ in sporotrichosis treatment. Further studies in experimental models are needed to understand the possible future application of this drug as an alternative therapy or as an adjuvant in sporotrichosis treatment., Lay Abstract: Sporotrichosis is a subcutaneous and lymphatic infection, caused by fungi of Sporothrix spp. Our study shows the potential of NikZ to inhibiting Sporothrix species in vitro. Further studies are needed to understand the future application of this drug to sporotrichosis treatment., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

We evaluated the growth and the susceptibility to oxidative stress of Sporothrix spp., exposed to different iron concentrations in culture medium, and the susceptibility of Sporothrix spp. to itraconazole, alone and in combination with to the iron chelator deferasirox. The results showed that the growth of S. brasiliensis isolates was more affected by iron availability in comparison to S. schenckii, but both fungal species conidia became more prone to oxidative stress when iron was added to culture medium. Conversely, the combination of itraconazole and deferasirox only resulted in synergism against a minority of S. schenckii isolates., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Sunitinib is a small molecule inhibitor of multiple receptor tyrosine kinases such as platelet derived growth factor receptor, vascular endothelial growth factor receptor, kit receptor and other receptors. The US Food and Drug Administration (FDA) has approved sunitinib for the treatment of advanced renal cell carcinoma and gastrointestinal stromal tumors. It has been reported that sunitinib was mainly metabolized by CYP3A but its pharmacokinetic interactions have not been revealed. In this study, we investigated whether CYP3A inhibitors (ketoconazole, voriconazole, and itraconazole) could influence the pharmacokinetics of sunitinib and its equipotent metabolite N-desethyl sunitinib in a drug-drug interaction study in Sprague Dawley (SD) rats. The results showed that ketoconazole and voriconazole significantly increased the exposure of sunitinib, decreased the exposure of N-desethyl sunitinib, and inhibited the metabolism of sunitinib in rats. However, itraconazole showed only a weak effect on pharmacokinetics and metabolism. Coadministration of sunitinib with ketoconazole and voriconazole should be avoided if possible or if not, there should be therapeutic drug monitoring of the levels of sunitinib and N-desethyl sunitinib. Therefore, drug-drug interaction should be considered when sunitinib is administered in conjunction with CYP3A inhibitors, which might lead to toxicity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Gene-treatment interactions, just like drug-drug interactions, can have dramatic effects on a patient response and therefore influence the clinician decision at the patient's bedside. Crossover designs, although they are known to decrease the number of subjects in drug-interaction studies, are seldom used in pharmacogenetic studies. We propose to evaluate, via realistic clinical trial simulations, to what extent crossover designs can help quantifying the gene-treatment interaction effect. We explored different scenarios of crossover and parallel design studies comparing two symptom-modifying treatments in a chronic and stable disease accounting for the impact of a one gene and one gene-treatment interaction. We varied the number of subjects, the between and within subject variabilities, the gene polymorphism frequency and the effect sizes of the treatment, gene, and gene-treatment interaction. Each simulated dataset was analyzed using three models: (i) estimating only the treatment effect, (ii) estimating the treatment and the gene effects, and (iii) estimating the treatment, the gene, and the gene-treatment interaction effects. We showed how ignoring the gene-treatment interaction results in the wrong treatment effect estimates. We also highlighted how crossover studies are more powerful to detect a treatment effect in the presence of a gene-treatment interaction and more often lead to correct treatment attribution., (© 2021 The Authors. CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Background: The antifungal drug itraconazole exerts in vitro activity against SARS-CoV-2 in Vero and human Caco-2 cells. Preclinical and clinical studies are required to investigate if itraconazole is effective for the treatment and/or prevention of COVID-19., Methods: Due to the initial absence of preclinical models, the effect of itraconazole was explored in a clinical, proof-of-concept, open-label, single-center study, in which hospitalized COVID-19 patients were randomly assigned to standard of care with or without itraconazole. Primary outcome was the cumulative score of the clinical status until day 15 based on the 7-point ordinal scale of the World Health Organization. In parallel, itraconazole was evaluated in a newly established hamster model of acute SARS-CoV-2 infection and transmission, as soon as the model was validated., Findings: In the hamster acute infection model, itraconazole did not reduce viral load in lungs, stools or ileum, despite adequate plasma and lung drug concentrations. In the transmission model, itraconazole failed to prevent viral transmission. The clinical trial was prematurely discontinued after evaluation of the preclinical studies and because an interim analysis showed no signal for a more favorable outcome with itraconazole: mean cumulative score of the clinical status 49 vs 47, ratio of geometric means 1.01 (95% CI 0.85 to 1.19) for itraconazole vs standard of care., Interpretation: Despite in vitro activity, itraconazole was not effective in a preclinical COVID-19 hamster model. This prompted the premature termination of the proof-of-concept clinical study., Funding: KU Leuven, Research Foundation - Flanders (FWO), Horizon 2020, Bill and Melinda Gates Foundation., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

P-glycoprotein (P-gp), is an important efflux pump involved in chemotherapy resistance in human colon cancer. We investigated the efficacy of itraconazole as a P-gp inhibitor and its therapeutic synergistic relationship to paclitaxel through 99m Tc-MIBI accumulation in HT-29 tumor-bearing nude mice. Histopathological screening along with in vitro experiments was done for further assessment. Itraconazole successfully inhibited P-gp mediated 99m Tc-MIBI efflux, increasing its in vitro accumulation in itraconazole-receiving dishes. Notably, the co-administration of itraconazole with paclitaxel significantly enhanced the in vitro cytotoxicity effect of paclitaxel in itraconazole + paclitaxel wells containing HT-29 cells. Compared to the control, tumor volume in mice treated with itraconazole, paclitaxel and itraconazole +paclitaxel showed growth suppression approximately by 36.21, 60.02, and 73.3% respectively. And compared to paclitaxel group, the nude mice co-treated with paclitaxel and itraconazole showed suppression of tumor growth by about 33.31 % at the end of the treatment period. Also the biodistribution result showed that the co-administration of itraconazole with paclitaxel raised the mean tumor radioactivity accumulation compared to control and paclitaxel group. When given paclitaxel alone, the ID% of hepatic and cardiac tissue was reduced while co-administration of itraconazole with paclitaxel increased 99m Tc-MIBI accumulation in these organs. Furthermore, the histopathological findings confirmed the biodistribution results. These results demonstrate that although monotherapy with itraconazole or paclitaxel has anti-tumor activity against HT-29 human colorectal cancer, a synergistic anti-tumor activity can be achieved when itraconazole is co-administered with paclitaxel. Also, 99m Tc-MIBI is an effective radiotracer for monitoring response to treatment in MDR tumors., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Sporotrichosis, a mycosis caused by pathogenic species of the genus Sporothrix, affects diverse species of mammals. Until 2007, Sporothrix schenckii was considered the unique etiologic agent of sporotrichosis. Canine sporotrichosis is a poorly reported disease, and the majority of cases are from Rio de Janeiro, Brazil. There are scarce studies on the characterization of canine isolates of Sporothrix schenckii complex, as well as few antifungal susceptibility data available. The aim of this study was to characterize the clinical isolates of Sporothrix from dogs from Brazil at species level and evaluate their antifungal susceptibility profile. Polyphasic taxonomy was used to characterization at species level (morphological, phenotypical characteristics, and molecular identification). Antifungal susceptibility profiles (amphotericin B, itraconazole, ketoconazole, posaconazole, and terbinafine) were determined using the Clinical and Laboratory Standards Institute broth microdilution method (M38-A2). According to phenotypic identification and molecular analysis, 46 isolates included in this study were identified as S. brasiliensis and one as S. schenckii. Amphotericin B presented the highest minimum inhibitory concentration values, and the other drugs showed effective in vitro antifungal activity. This is the first report of S. schenckii in dogs from Brazil, since S. brasiliensis is the only species that has been described in canine isolates from Rio de Janeiro to date. Nevertheless, no differences were observed in the antifungal susceptibility profiles between the S. brasiliensis and S. schenckii isolates, and it is important to continuously study new canine clinical isolates from Rio de Janeiro, Brazil.

Sporotrichosis is one of the neglected tropical diseases causing subcutaneous chronic granulomatous lesion by thermally dimorphic fungi belonging to Sporothrix species. Sporothrix brasiliensis, Sporothrix mexicana and Sporothrix globosa are the common pathogenic species. In Asian countries, S. globosa constitutes nearly 99.3% of all Sporothrix species. We studied 63 cases of sporotrichosis of geographically diverse origin from India and Sporothrix isolates were characterised for its growth in different media, temperatures, ability to assimilate sugars and antifungal susceptibility profile. Molecular characterization was performed by sequencing of the calmodulin (CAL), beta tubulin (BT) and translational elongation factor 1-alpha (TEF-1α) and typing by fluorescent amplified fragment length polymorphism (FAFLP). In patients who presented with fixed (49.2%), lymphocutaneous lesions (23.8%), in 26.9% the details were not known, none had systemic dissemination. All the isolates tested were Sporothrix globosa and that could grow up to 35 °C and unable to grow at and beyond 37 °C. The assimilation of sucrose, ribitol and raffinose helps in identifying S. globosa. Sequences of CAL or BT or TEF-1α can differentiate S. globosa from other species in the complex. FAFLP results exhibited low genetic diversity. No correlation was noted between genotypes and clinical presentation, or geographic distribution. Itraconazole, terbinafine and posaconazole showed good in vitro antifungal activity against S. globosa whereas fluconazole and micafungin had no activity. S. globosa of Indian origin is relatively less pathogenic than other pathogenic Sporothrix species as it does not cause systemic dissemination and in the diagnostic laboratory, incubation of the cultures below 37 °C is essential for effective isolation.

Fonsecaea monophora, which is very similar to Fonsecaea pedrosoi in morphological features, has been commonly misdiagnosed as F. pedrosoi. Like F. pedrosoi, F. monophora has been also identified as a predominant pathogen of Chromoblastomycosis (CBM). Melanin has been recognized as a virulence factor in several fungi, however, it is still largely unknown about the biological role of melanin and how melanin is synthesized in F. monophora. In this study, we identified two putative polyketide synthase genes (pks), AYO21&#95;03016 (pksA) and AYO21&#95;10638, by searching against the genome of F. monophora. AYO21&#95;03016 and AYO21&#95;10638 were further targeted disrupted by Agrobacterium tumefaciens-mediated transformation (ATMT). We discovered that pksA gene was the major polyketide synthase required for melanin synthesis in F. monophora, rather than AYO21&#95;10638. Phenotypic analysis showed that, knocking out of the pksA gene attenuated melanogenesis, growth rate, sporulation ability and virulence of F. monophora, as compared with wild-type and complementation strain (pksA-C). Furthermore, the ΔpksA mutant was confirmed to be more sensitive to the oxidative stress, extreme pH environment, and antifungal drugs including itraconazole (ITC), terbinafine (TER), and amphotericin B (AMB). Taken together, these findings enabled us to comprehend the role of pksA in regulating DHN-melanin pathway and its effect on the biological function of F. monophora., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Itraconazole is the first drug of choice for the treatment of sporotrichosis and it is available at different concentrations for veterinary patients. However, therapeutic failure has been reported, limiting clinical treatment. This study evaluated the in vitro efficacy of brand-name and compounded itraconazole formulations against Sporothrix brasiliensis and estimated the itraconazole content in each tested formulation. Oral capsules were acquired from two brand-name products for human (H-IND) and veterinary (V-IND) uses, and three from compounding pharmacies in Pelotas, RS, for human (H-COMP1/H-COMP2) and veterinary (V-COMP) uses. Capsule purity was analyzed by liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS). Antifungal activity was determined against 29 Sporothrix brasiliensis by the M38-A2 guideline of CLSI. H-IND/H-COMP1/H-COMP2 had high efficacy against S. brasiliensis (approximately 70% of total isolated susceptible), V-COMP showed moderate efficacy (51.7%), and V-IND was the least effective formulation (37.9%). Thirty-four percent of the total isolates were resistant to all formulations. Furthermore, itraconazole content did not match the concentration indicated by the manufacturers, ranging from 387.70 to 7.81 μg/mg (H-COMP2 > V-COMP > H-IND > H-COMP1 > V-IND). Therefore, it is possible that the formulations showed different in vitro efficacy due to the difference in their itraconazole contents. Given the emergence of antifungal resistance for all formulations, the choice product to be used must follow susceptibility testing. Stringent quality control measures are recommended for product manufactures to assure drug content uniformity.

The dimorphic fungus Sporothrix globosa is the predominant etiologic agent causing sporotrichosis in China, particularly in the northeast. It has been demonstrated that the incubation temperature and growth phase can influence in vitro antifungal susceptibility profiles of S. schenckii sensu stricto and S. brasiliensis (sibling species of S. globosa). Few studies have reported on the antifungal susceptibility of S. globosa, especially using large numbers of isolates. In this study, we assessed the susceptibility of 80 isolates of S. globosa originating from Jilin Province, northeastern China, to six antifungal agents (itraconazole, terbinafine, voriconazole, posaconazole, fluconazole, and amphotericin B), at varying incubation temperatures and in different fungal growth phases. The isolates were most sensitive to terbinafine (geometric mean [GM] of the minimum inhibitory concentration [MIC]: 0.0356 μg/ml for the mycelial phase at 30 °C, 0.0332 μg/ml for the mycelial phase at 35 °C, and 0.031 μg/ml for the yeast phase, respectively), followed by posaconazole (GM of the MIC: 4.2501 μg/ml for the mycelial phase at 30 °C, 1.4142 μg/ml for the mycelial phase at 35 °C, and 0.7195 μg/ml for the yeast phase, respectively) and itraconazole (GM of the MIC: 6.8448 μg/ml for the mycelial phase at 30 °C, 3.1383 μg/ml for the mycelial phase at 35 °C, and 1.0263 μg/ml for the yeast phase, respectively). The isolates were relatively resistant to fluconazole (GM of the MIC: 76.7716 μg/ml for the mycelial phase at 30 °C, 66.2570 μg/ml for the mycelial phase at 35 °C, and 24.4625 μg/ml for the yeast phase, respectively) and voriconazole (GM of the MIC: 26.2183 μg/ml for the mycelial phase at 30 °C, 13.6895 μg/ml for the mycelial phase at 35 °C, and 1.3899 μg/ml for the yeast phase, respectively). For all the tested azole drugs, the MICs at 30 °C were significantly higher than those at 35 °C (P < 0.001); for all agents except terbinafine, the MICs of S. globosa in the yeast phase were significantly lower than those of the strains in the mycelial phase (P < 0.001). These results show that the sensitivities of S. globosa to antifungal compounds are dependent on incubation temperature and growth phase. To the best of our knowledge, this is the largest study of antifungal susceptibility of S. globosa isolates reported to date. To establish epidemiological cutoff values for S. globosa, further antifungal susceptibility testing studies by independent laboratories located in different regions and using uniform conditions are required.

There are only a few combinations of antifungal drugs with known resistance marker genes in the Aspergillus species; therefore, the transformation of their wild-type strains is limited. In this study, to develop the novel dominant selectable marker for itraconazole, a fungal cell membrane synthesis inhibitor, we focused on Aspergillus luchuensis cyp51A (Alcyp51A), which encodes a 14-α-sterol demethylase related to the steroid synthesis pathway. We found that the G52R mutation in AlCyp51A and the replacement of the native promoter with a high-expression promoter contributed to itraconazole resistance in Aspergillus oryzae, designated as itraconazole resistant gene (itrA). The random integration in the A. luchuensis genome of the itrA marker cassette gene also allowed for transformation using itraconazole. Therefore, we succeed in developing a novel itraconazole resistance marker as a dominant selectable marker for transformation in A. oryzae and A. luchuensis., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

We present an in vitro susceptibility assay for Madurella mycetomatis hyphae using resazurin for endpoint reading. Using this assay, reproducible MICs were obtained for amphotericin B, itraconazole, voriconazole, posaconazole, terbinafine, and micafungin. Results were comparable with those of a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2 H -tetrazolium-5-carboxanilide salt (XTT)-based susceptibility assay. The lowest MICs were obtained for itraconazole and posaconazole (MIC 50 , 0.016 µg/ml) followed by voriconazole (MIC 50 , 0.063 µg/ml). Amphotericin B, micafungin, and terbinafine appeared much less effective., (Copyright © 2021 Abd Algaffar et al.)

Radiotherapy is a common therapy method for nasopharyngeal carcinoma (NPC) treatment; however, radioresistance greatly limits the clinical efficiency and prognosis of NPC patients. Therefore, it is extremely urgent to reveal the underlying mechanism contributing to radioresistance and find possible diagnostic biomarkers. Here, we collected the spheroids formed by NPC cells, which had been confirmed to hold the stem cell-like traits, and found that these spheroids exhibited a certain degree of radioresistance. Additionally, NPC spheroids displayed a certain degree of ferroptosis resistance, as evident by the decrease of iron concentration in lysosomes and lipid peroxides oxygen, and increase of glutathione (GSH) level. Furthermore, we revealed that itraconazole triggered the ferroptosis of NPC spheroids, which is characterized as the increase of iron concentration and lipid peroxides oxygen, and decrease of GSH level, and decreased the cell viability of NPC spheroids. Notably, itraconazole partially reversed the radioresistance of NPC spheroids. Mechanistically, we found that itraconazole can sequester iron in lysosome and thus trigger ferroptosis; this is essential for itraconazole-mediated attenuation on NPC spheroid stemness. Therefore, this study provides evidences showing that itraconazole might be used for killing NPC stem cells and thus attenuate radioresistance., (© 2020 Wiley Periodicals LLC.)

Over the past 20-30 years, Trichophyton rubrum represented the most widespread dermatophyte with a prevalence accounting for 70% of dermatophytosis. The treatment for cutaneous infections caused by Trichophyton spp. are imidazoles (ketoconazole (KTZ)) and triazoles (itraconazole (ITZ)). T. rubrum can develop resistance to azoles after prolonged exposure to subinhibitory concentrations resulting in therapeutic failures and chronic infections. These problems have stimulated the search for therapeutic alternatives, including essential oils, and their potential use in combination with conventional antifungals. The purpose of this study was to evaluate the antifungal activity of tea tree oil (TTO) ( Melaleuca alternifolia essential oil) and the main components against T. rubrum and to assess whether TTO in association with KTZ/ITZ as reference drugs improves the antifungal activity of these drugs. We used a terpinen-4-ol chemotype (35.88%) TTO, and its antifungal properties were evaluated by minimum inhibitory and minimum fungicidal concentrations in accordance with the CLSI guidelines. The interaction between TTO and azoles was evaluated through the checkerboard and isobologram methods. The results demonstrated both the fungicide activity of TTO on T. rubrum and the synergism when it was used in combination with azoles. Therefore, this mixture may reduce the minimum effective dose of azole required and minimize the side effects of the therapy. Synergy activity offered a promise for combination topical treatment for superficial mycoses.

Aims: According to the WHO, 20-25% of people worldwide are affected by skin infections caused by dermatophytes, such as those of the Trichophyton genus. Additionally, several dermatophytes have developed resistance to drugs such as griseofulvin and itraconazole. This study tested 2S albumins-derived antimicrobial peptides (AMPs) as alternative antidermatophytic molecules., Main Methods: Membrane pore formation assays, tests to detect overproduction of ROS, scanning electron microscopy (SEM) and fluorescence microscopy (FM) were carried out to provide insight into the mechanisms of antidermatophytic action., Key Findings: All AMPs (at 50 μg mL -1 ) tested reduced the mycelial growth of T. mentagrophytes and T. rubrum by up to 95%. In contrast, using a concentration 20-fold higher, griseofulvin only inhibited T. mentagrophytes by 35%, while itraconazole was not active against both dermatophytes. Scanning electron and fluorescence microscopies revealed that the six AMPs caused severe damage to hyphal morphology by inducing cell wall rupture, hyphal content leakage, and death. Peptides also induced membrane pore formation and oxidative stress by overproduction of ROS. Based on the stronger activity of peptides than the commercial drugs and the mechanism of action, all six peptides have the potential to be either employed as models to develop new antidermatophytic drugs or as adjuvants to existing ones., Significance: The synthetic peptides are more efficient than conventional drug to treat infection caused by dermatophytes being potential molecules to develop new drugs., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A four-month old female Okinawa rail (Hypotaenidia okinawae) presented with respiratory distress. Despite antifungal treatment with voriconazole (VRZ), micafungin (MCF), and itraconazole (ITZ), respiratory distress did not improve and the bird died 167 days after initiating treatment. Necropsy revealed multifocal pyogranulomatous necrotic nodular lesions with numerous whitish-green fungal hyphae in the left air sac. Aspergillus flavus was isolated from the left air sac lesion. Antifungal susceptibility tests indicated that the isolate showed low susceptibility to amphotericin B (AMB), fluconazole (FLZ), VRZ and MCF.