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Background: As a rare subcutaneous infection, protothecosis is easily misdiagnosed. Similar to other subcutaneous infection, there is no unified standard for treatment, for cases not suitable for surgery, clinicians often use antifungal drugs based on their experience, and the course of treatment varies from several months to several years. Based on the fact that there are few relevant materials and researches on photodynamic therapy (PDT), we conducted a study based on a clinical case that used oral itraconazole combined with 5-aminolevylinic acid photodynamic therapy (ALA-PDT) to treat a patient with cutaneous protothecosis caused by Prototheca wicherhamii., Methods: Different concentrations of ALA and different light doses were used to investigate the effects of ALA-PDT on the growth inhibition of P. wickerhamii in vitro with Colony-counting Methods. And we used transmission electron microscopy (TEM) to visualize the structural changes and the effects of ALA-PDT treating on cellular structures of the P. wickerhamii. Futher, we performed the susceptibility test of P. wickerhamii to itraconazole before and after ALA-PDT in vitro., Results: We have successfully treated a patient with cutaneous protothecosis caused by P. wickerhamii by using combination therapy in a total of 9-week course of treatment. In vitro, ALA-PDT can inhibit the growth of P. wickerhamii when the ALA concentration was 5 mg/mL (P < 0.01), and this effect became stronger as the concentration of ALA or light dose is increased. Using TEM, we confirmed that ALA-PDT can disrupt the cell wall structure and partition structure of P. wickerhamii, which may contribute to its inhibitory effect. Further studies showed that the MIC of itraconazole for P. wickerhamii was decreased after ALA-PDT., Conclusions: ALA-PDT combined with oral itraconazole can be used to treat cutaneous protothecosis. Accordingly, ALA-PDT can destroy the cell wall and partition structure of P. wickerhamii leading to an inhibitory effect on it in vitro, and the effect is enhanced with the increase of ALA concentration and light dose. Also, the sensitivity of P. wickerhamii to itraconazole is observed increased after ALA-PDT. So our study provides a theoretical basis for the promising treatment against cutaneus protothecosis., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Sorafenib is a multikinase inhibitor employed for managing hepatocellular carcinoma (HCC). The emergence of sorafenib resistance presents an obstacle to its therapeutic efficacy. One notable approach to overcoming sorafenib resistance is the exploration of combination therapies. The role of hedgehog signaling in sorafenib resistance has been also examined in HCC. R51211, known as itraconazole, has been safely employed in clinical practice. Through in vitro and in vivo investigations, we assessed the potential of R51211 to enhance the therapeutic efficacy of sorafenib by inhibiting the hedgehog signaling. The zero-interaction potency synergy model demonstrated a synergistic interaction between R51211 and sorafenib, a phenomenon reversed by the action of a smoothened receptor agonist. This dual therapy exhibited an increased capacity to induce apoptosis, as evidenced by alterations in the Bax/BCL-2 ratio and caspase-3, along with a propensity to promote autophagy, as indicated by changes in BECN1, p62, and the LC3I/LC3II ratio. Furthermore, the combination therapy resulted in significant reductions in biomarkers associated with liver preneoplastic alterations, improved liver microstructure, and mitigated changes in liver function enzymes. The substantial decrease in hedgehog components (Shh, SMO, GLI1, and GLI2) following R51211 treatment appears to be a key factor contributing to the increased efficacy of sorafenib. In conclusion, our study highlights the potential of R51211 as an adjunct to sorafenib, introducing a new dimension to this combination therapy through the modulation of the hedgehog signaling pathway. Further investigations are essential to validate the therapeutic efficacy of this combined approach in inhibiting the development of liver cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The CUSP9 protocol is a polypharmaceutical strategy aiming at addressing the complexity of glioblastoma by targeting multiple pathways. Although the rationale for this 9-drug cocktail is well-supported by theoretical and in vitro data, its effectiveness compared to its 511 possible subsets has not been comprehensively evaluated. Such an analysis could reveal if fewer drugs could achieve similar or better outcomes. We conducted an exhaustive in vitro evaluation of the CUSP9 protocol using COMBImageDL, our specialized framework for testing higher-order drug combinations. This study assessed all 511 subsets of the CUSP9v3 protocol, in combination with temozolomide, on two clonal cultures of glioma-initiating cells derived from patient samples. The drugs were used at fixed, clinically relevant concentrations, and the experiment was performed in quadruplicate with endpoint cell viability and live-cell imaging readouts. Our results showed that several lower-order drug combinations produced effects equivalent to the full CUSP9 cocktail, indicating potential for simplified regimens in personalized therapy. Further validation through in vivo and precision medicine testing is required. Notably, a subset of four drugs (auranofin, disulfiram, itraconazole, sertraline) was particularly effective, reducing cell growth, altering cell morphology, increasing apoptotic-like cells within 4-28 h, and significantly decreasing cell viability after 68 h compared to untreated cells. This study underscores the importance and feasibility of comprehensive in vitro evaluations of complex drug combinations on patient-derived tumor cells, serving as a critical step toward (pre-)clinical development., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest to disclose., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: Due to the potential for Aspergillus species to cause lethal infections and the rising rates of antifungal resistance, the significance of antifungal susceptibility tests has increased. We aimed to assess the sensitivities of Aspergillus species to amphotericin B (AMB), voriconazole (VOR), itraconazole (ITZ), and caspofungin (CAS) using disk diffusion (DD) and gradient diffusion (GD) methods and compare them with broth microdilution (BMD) as the reference susceptibility method., Methods: The study involved 62 Aspergillus fumigatus, 28 Aspergillus flavus, and 16 Aspergillus terreus isolates, totaling 106 Aspergillus isolates. BMD and DD methods were performed in accordance with CLSI M38-A2 and CLSI M51-A documents, respectively. The GD method utilized nonsupplemented Mueller Hinton agar (MHA) as the medium., Results: In the BMD method, the lowest minimal inhibitory concentration (MIC) 90 or minimal effective concentration (MEC) 90 values were observed for VOR and CAS (0.5 μg/mL and 0.06 μg/mL, respectively). AMB and ITZ MIC 90 values were both 2 μg/mL. In our comparison of the GD method with the BMD method at ±2 dilution, we observed essential agreement rates of 91.6%, 99.1%, 100%, and 38.6% for AMB, VOR, ITZ, and CAS, respectively. When comparing DD and BMD methods, we found categorical agreement rates of 65.1%, 99.1%, 77.3%, and 100% for AMB, VOR, ITZ, and CAS, respectively. For GD and BMD methods, these rates were 79.2%, 99.1%, 87.8%, and 100%., Conclusions: Given the high essential and categorical agreement rates, we posit that the GD method is a viable alternative to the BMD method for AMB, ITZ and VOR but not for CAS. In addition, the use of nonsupplemented MHA in the GD method proves advantageous due to its cost-effectiveness and widespread availability compared to other growth media., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

Chitosan has received much more attention as a functional biopolymer with applications in pharmaceuticals, agricultural, drug delivery systems and cosmetics. The objectives of present investigation were to carry out modification of chitosan for enhancement of aqueous solubility, which will impart increased solubility and dissolution rate of poorly soluble drug itraconazole (ITZ) and also evaluate the modified chitosan for soyabean seed germination studies. The modification of chitosan was accomplished through the antisolvent precipitation method; employing five carboxylic acids. The resulting products were assessed for changes in molecular weight, degree of deacetylation, solubility and solid state characterization. Subsequently, the modified chitosan was complexed with itraconazole using the co-grinding technique. The prepared formulations were evaluated for solubility, FTIR (Fourier-transform infrared spectroscopy), PXRD (Powder X-ray diffraction), in-vitro dissolution studies. Furthermore the effect of modified chitosan has been evaluated on soybean seed germination. Results demonstrated that, modified chitosan improves self and solubility of itraconazole by six folds. As there was increased degree of deacetylation of chitosan leads to improvement in solubility. The results of FTIR showed the slight shifting of peaks in co-grind formulations of itraconazole. Formulations showed reduction in crystallinity of drug which leads to enhancement in dissolution rate as compared to pure itraconazole. Retention of property of seed germination was observed with modified chitosan at optimum concentration of 3 % w/v, with benefit of enhanced aqueous solubility of chitosan. This positive result paves the way for the advancement of pharmaceutical and agrochemical products employing derivatives of chitosan., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: To determine the epidemiological cut-off values (ECVs) of ten antifungal agents in a wide range of yeasts and Aspergillus spp. using gradient concentration strips., Methods: The minimum inhibitory concentrations for amphotericin B, anidulafungin, caspofungin, micafungin, flucytosine, fluconazole, itraconazole, isavuconazole, posaconazole, and voriconazole, determined with gradient concentration strips at 35 French microbiology laboratories between 2002 and 2020, were retrospectively collected. Then, the ECVs were calculated using the iterative method and a cut-off value of 97.5%., Results: Minimum inhibitory concentrations were available for 17 653 clinical isolates. In total, 48 ECVs (including 32 new ECVs) were determined: 29 ECVs for frequent yeast species (e.g. Candida albicans and itraconazole/flucytosine, and Candida glabrata species complex [SC] and flucytosine) and rare yeast species (e.g. Candida dubliniensis, Candida inconspicua, Saccharomyces cerevisiae, and Cryptococcus neoformans) and 19 ECVs for Aspergillusflavus SC, Aspergillusfumigatus SC, Aspergillusnidulans SC, Aspergillusniger SC, and Aspergillusterreus SC., Conclusions: These ECVs can be added to the already available gradient concentration strip-specific ECVs to facilitate minimum inhibitory concentration interpretation and streamline the identification of nonwild type isolates., (Copyright © 2022 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

This study aimed to evaluate the miscibility of cellulose derivatives to improve the release rate and stability of microparticles containing the weakly basic drug itraconazole (ITZ). We also investigated the effect of some organic acids on the microenvironmental pH (pH m ) and the release rate of ITZ from the cellulose-based microparticles. The synergistic effect of cellulose-based microparticles and pH m modulators on the bioavailability of ITZ compared with the reference product was investigated in a rabbit model. Differential scanning calorimetry and Fourier-transform infrared spectroscopy (FTIR) analysis showed that ITZ, hydroxypropyl methylcellulose, and hydroxypropyl methylcellulose phthalate were miscible at a ratio of 1.5:3:1 (w/w/w), and the stability of the microparticles was maintained for 6 months under accelerated conditions. In addition, X-ray diffraction, FTIR, and scanning electron microscopy were used to characterize the properties of the microparticles. Through the titration technique and determination of pH m , the combination of fumaric acid and maleic acid (1:2, w/w) was found to be the most effective pH m modulator for microparticles. The integration of cellulose-based microparticles and pH m modulators showed a synergistic effect on the flux and relative bioavailability of ITZ and its active metabolite OH-ITZ (182.60 % and 217.67 %, respectively) when compared with the reference product., Competing Interests: Declaration of competing interest The authors do not have any personal conflict of interest that may arise from submission of our research., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Paracoccidioidomycosis (PCM) caused by Paracoccidioides brasiliensis (Pb), is a severe mycosis, prevalent in tropical countries. The presence of polymorphonuclear neutrophils (PMN) in lesions is conspicuous, indicating their central role in innate immunity through the direct killing of Pb and the production of cytokines that activate acquired immunity in the presence of itraconazole (Itra). The toxicity and direct antifungal activity of Itra on Pb in splenocyte co-cultures were evaluated in vitro. Itra showed no toxic effect but marked antifungal activity against Pb. Purified PMN were obtained by the subcutaneous (SC) injection of Pb into mice. Results showed the effect of Itra on the size of the air pouch produced, the cellular population that migrated to the infection site, protein, and mitochondrial metabolism patterns, production of ROS an NO, and the number of cytokines synthesized. Lower doses (3 and 10 mg/kg) of Itra did not affect the cellular profile but led to a lower influx of viable more active PMN, and increased production of ROS and proteins. At a dose of 50 mg/kg the PMN profile remained unchanged along with all other parameters analyzed remained unaltered. Decreases in most cytokine levels were inversely proportional to the Itra concentration. Lower Itra concentrations may elicit activation of the immune response because the combined effects of therapy and immune response are needed, while the higher dose does not require it. Itra also promotes the activation of the cytokines which elicit PMN activation and consequently the resolution of Pb18 infection in the air pouch., Competing Interests: Declaration of competing interest All authors declare that they have no conflict of interest., (Published by Elsevier Inc.)

Background and Aims: Bleomycin (BLM) is one of the antitumor medications that had proven efficacy in the treatment of a wide range of malignant conditions. Pulmonary fibrosis which is frequently encountered during the course of bleomycin therapy may significantly reduce the potential efficacy of bleomycin in cancer therapy. This study tested the hypothesis that itraconazole may have mitigating effects on BLM-induced pulmonary fibrosis and tried to delineate the potential mechanisms of these effects., Materials and Methods: In a rat model of pulmonary fibrosis elicited by BLM, the effect of different doses of itraconazole was explored at the biochemical, histopathological, and electron microscopic levels., Key Findings: Itraconazole, in a dose-dependent manner, exhibited significant effects on the pro-oxidant/antioxidant balance, the inflammatory consequences, high-mobility group box 1/toll-like receptor-4 Axis, autophagy and nuclear factor kappa B/Nod-like receptor protein 3 inflammasome signaling and alleviated the histopathological, immunohistochemical, and electron microscopic perturbations induced by BLM in the pulmonary tissues., Significance: In view of the afore-mentioned data, itraconazole may be a promising drug that efficiently mitigates the deleterious effects of BLM on the pulmonary tissues., Competing Interests: Declaration of competing interest No conflict of interest was declared by the authors. This research didn’t receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Fighting malignant neoplasms via repurposing existing drugs could be a welcome move for prosperous cancer remediations. In the current work, nanovehiculation and optimization of the repositioned itraconazole (ITZ) utilizing ascorbyl palmitate (AP) aspasomes would be an auspicious approach. Further, the optimized aspasomes were incorporated in a cream and tracked for skin deposition. The in vivo efficacy of aspasomal cream on mice subcutaneous Ehrlich carcinoma model was also assessed. The optimized aspasomes revealed nano size (67.83 ± 6.16 nm), negative charge (-79.40 ± 2.23 mV), > 95% ITZ entrapment and high colloidal stability. AP yielded substantial antioxidant capacity and pushed the ITZ cytotoxicity forward against A431 cells (IC 50 = 5.3±0.27 μg/mL). An appealing privilege was the aspasomal cream that corroborated spreadability, contemplated skin permeation and potentiated in vivo anticancer competence, reflected in 62.68% reduction in the tumor weight. Such synergistic tumor probes set the foundation for futuristic clinical translation and commercialization., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Introduction: Trichophyton rubrum is an anthropophilic dermatophyte that is most frequently isolated from onychomycosis (tinea unguium) worldwide. T. rubrum strains showing resistance to the anti-fungal drug terbinafine (TRF) have also been isolated from human patients worldwide., Methods: In this study, we isolated a TRF-resistant strain (N99-2) of T. rubrum from a patient with recurrent tinea unguium. In vitro susceptibility of the clinical isolate to TRF, itraconazole (ITZ), ravuconazole (RVZ), and luliconazole (LCZ) was investigated using the Clinical & Laboratory Standards Institute M38-A2 test. To identify mutations, we compared the gene sequence of N99-2 to that of a TRF-susceptible strain of T. rubrum. Results; In N99-2, the minimum inhibitory concentrations were 32 mg/L for TRF, <0.03 mg/L for ITZ, <0.03 mg/L for RVZ, and <0.03 mg/L for LCZ. The squalene epoxidase (SQLE) gene sequence in N99-2 was determined to be 1467 bp in length, and it encoded a protein of 488 amino acids, beginning with a putative initiating methionine (ATG). The following mutations were identified from the SQLE of N99-2: L393F and Y394del., Conclusions: This is the first report of the detection of a deletion mutation in SQLE in a TRF-resistant strain. The protein of SQLE is the target of TRF, and it is essential for cell membrane synthesis in dermatophytes. However, dermatophyte cells were found to undergo gene mutations to escape the effects of antifungal agents., (Copyright © 2022 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

The prevalence of invasive aspergillosis with azole resistance is increasing, but the mechanisms underlying the development of resistance and treatment strategies are still limited. The present work is focused on finding a relationship between long-chain unsaturated fatty acids (LCUFAs), Aspergillus fumigatus development, and antifungal resistance. The effects of LCUFAs on antifungal agents in vitro were determined, and the stearic acid desaturase gene (sdeA) of A. fumigatus was characterized. In in vitro antifungal tests, LCUFAs antagonized the antifungal activity of itraconazole by extracting it from media, thereby preventing it from entering cells. The OA auxotrophic phenotype caused by an sdeA deletion confirmed that SdeA was required for OA biosynthesis in A. fumigatus. Furthermore, several low-level sdeA-overexpressing mutants with impaired vegetative growth phenotypes were successfully constructed. Additionally, an sdeA-overexpressing mutant, OEsdeA-5, showed lowered sensitivity levels to itraconazole. Moreover, RNA sequencing of OEsdeA-5 revealed that the altered gene-expression pattern. Through targeted metabolomics, decreased palmitic acid and stearic acid contents, accompanied by higher palmitoleic acid, margaroleic acid, and OA production levels, were found in OEsdeA-5. This study provides a novel insight of understanding of azole resistance and a potential target for drug development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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.)

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.)

Background: EUCAST has revised the definition of the susceptibility category I from 'Intermediate' to 'Susceptible, Increased exposure'. This implies that I can be used where the drug concentration at the site of infection is high, either because of dose escalation or through other means to ensure efficacy. Consequently, I is no longer used as a buffer zone to prevent technical factors from causing misclassifications and discrepancies in interpretations. Instead, an Area of Technical Uncertainty (ATU) has been introduced for MICs that cannot be categorized without additional information as a warning to the laboratory that decision on how to act has to be made. To implement these changes, the EUCAST-AFST (Subcommittee on Antifungal Susceptibility Testing) reviewed all, and revised some, clinical antifungal breakpoints., Objectives: The aim was to present an overview of the current antifungal breakpoints and supporting evidence behind the changes., Sources: This document is based on the ten recently updated EUCAST rationale documents, clinical breakpoint and breakpoint ECOFF documents., Content: The following breakpoints (in mg/L) have been revised or established for Candida species: micafungin against C. albicans (ATU = 0.03); amphotericin B (S ≤/> R = 1/1), fluconazole (S ≤/> R = 2/4), itraconazole (S ≤/> R = 0.06/0.06), posaconazole (S ≤/> R = 0.06/0.06) and voriconazole (S ≤/> R = 0.06/0.25) against C. dubliniensis; fluconazole against C. glabrata (S ≤/> R = 0.001/16); and anidulafungin (S ≤/> R = 4/4) and micafungin (S ≤/> R = 2/2) against C. parapsilosis. For Aspergillus, new or revised breakpoints include itraconazole (ATU = 2) and isavuconazole against A. flavus (S ≤/> R = 1/2, ATU = 2); amphotericin B (S ≤/> R = 1/1), isavuconazole (S ≤ /> R = 1/2, ATU = 2), itraconazole (S ≤/> R = 1/1, ATU = 2), posaconazole (ATU = 0.25) and voriconazole (S ≤/> R = 1/1, ATU = 2) against A. fumigatus; itraconazole (S ≤/> R = 1/1, ATU = 2) and voriconazole (S ≤/> R = 1/1, ATU = 2) against A. nidulans; amphotericin B against A. niger (S ≤/> R = 1/1); and itraconazole (S ≤/> R = 1/1, ATU = 2) and posaconazole (ATU = 0.25) against A. terreus., Implications: EUCAST-AFST has released ten new documents summarizing existing and new breakpoints and MIC ranges for control strains. A failure to adopt the breakpoint changes may lead to misclassifications and suboptimal or inappropriate therapy of patients with fungal infections., (Copyright © 2020. Published by Elsevier Ltd.)

Aims: To investigate the therapeutic potential of itraconazole in oral squamous cell carcinoma (OSCC) and its molecular mechanism., Materials and Methods: The in vitro anti-cancer effects of itraconazole was determined by CCK-8 assay and colony formation assay. Transwell and wound healing assays were used to examine cell invasion and migration. The in vivo therapeutic efficacy of itraconazole was assessed by OSCC patient-derived xenograft (PDX) model. Western blot was performed to explore the anti-cancer mechanism., Key Findings: Itraconazole inhibited cell proliferation and colony formation of OSCC cells in a time and concentration dependent manner; induced cell cycle arrest and apoptosis, as well as inhibited cell invasion and migration. In the OSCC PDX model, itraconazole impeded tumor growth, reduced Ki-67 expression and induced apoptosis. Itraconazole downregulated the protein expression of Hedgehog pathway to inhibit proliferation and migration of oral squamous cell carcinoma cells, which can be revised by recombinant human sonic hedgehog protein (rSHH)., Significance: Itraconazole showed anti-cancer effects on OSCC via inhibiting the Hedgehog pathway., Competing Interests: Declaration of competing interest The authors declared that there are no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Loss of stability of pharmaceutical APIs (Active Pharmaceutical Ingredient) due to photolytic activity has been a major concern in the pharmaceutical industry as it leads to loss of activity of API and excipients, the formation of toxic by-products, change in color and flavor. Itraconazole (ITZ) bulk drug was exposed to UVC (254 nm) irradiation in an environmental chamber (37 °C, 75 %RH) and its photoprotection by cinnamon, clove, eugenol, and oregano based microemulsion was analyzed. No significant change in the spectra was observed at various time points, confirming the photo-protective activity of microemulsions, unlike the bulk drug. FTIR spectra illustrate the fundamental peaks of the functional groups of ITZ and ITZ loaded MEs. The overlaid spectra showed that there was a minor change in peaks of UV exposed ITZ bulk drug but the ITZ loaded microemulsions were able to protect all the major functional groups. The in vitro anti-microbial assay against C. albicans demonstrated no significant change in the activity of ITZ loaded microemulsion between untreated, 7th day and 15th day while the activity of bulk drug was reduced drastically in the UVC exposed sample. It was concluded that microemulsions can be used as an effective photo-protective drug delivery vehicle for light-sensitive compounds., Competing Interests: Declaration of Competing Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Itraconazole (ITCZ) is used to treat pulmonary aspergillosis, but findings regarding the range of effective plasma concentrations are often contradictory. This study attempted to determine effective plasma concentrations of ITCZ and its active metabolite hydroxyitraconazole (OH-ITCZ) by retrospectively analyzing their relationships to clinical efficacy., Methods: The study included 34 patients with pulmonary aspergillosis treated using ITCZ (mean age, 70 years). Each patient was treated with 200 mg ITCZ once daily (mean duration of treatment: 384 days). Plasma concentrations of ITCZ and OH-ITCZ at trough levels from 7 to 889 days after the start of treatment were determined using high-performance liquid chromatography. Clinical efficacy was assessed through the improvement clinical symptoms., Results: Fifteen patients were classified as effective group and the other 19 patients as non-effective group. Mean (±standard deviation) ITCZ trough plasma concentration was significantly higher in effective group (1254 ± 924 ng/mL) than in non-effective group (260 ± 296 ng/mL). Mean OH-ITCZ plasma concentration was significantly higher in effective group (1830 ± 1031 ng/mL) than in non-effective group (530 ± 592 ng/mL). Receiver operating characteristic curve analysis revealed the optimal cutoff for ITCZ trough plasma concentration was 517 ng/mL, and 86.7% of effective group showed concentrations exceeding this value. The optimal cutoff for total ITCZ + OH-ITCZ plasma concentration was 1025 ng/mL, and 93.3% of effective group showed a concentration exceeding this value., Conclusions: Our findings indicate that effective plasma concentration ranges for the treatment of pulmonary aspergillosis begin at an ITCZ trough plasma concentration of 500 ng/mL and a total ITCZ + OH-ITCZ plasma concentration of 1000 ng/mL., (Copyright © 2019. Published by Elsevier Ltd.)

Plant proteins are emerging as an alternative to conventional treatments against candidiasis. The aim of this study was to better understand the mechanism of action of Mo-CBP 2 against Candida spp, evaluating redox system activity, lipid peroxidation, DNA degradation, cytochrome c release, medium acidification, and membrane interaction. Anti-candida activity of Mo-CBP 2 decreased in the presence of ergosterol, which was not observed with antioxidant agents. C. albicans treated with Mo-CBP 2 also had catalase and peroxidase activities inhibited, while superoxide dismutase was increased. Mo-CBP 2 increased the lipid peroxidation, but it did not alter the ergosterol profile in live cells. External medium acidification was strongly inhibited, and cytochrome c release and DNA degradation were detected. Mo-CBP 2 interacts with cell membrane constituents, changes redox system enzymes in C. albicans and causes lipid peroxidation by ROS overproduction. DNA degradation and cytochrome c release suggest apoptotic or DNAse activity. Lipid peroxidation and H + -ATPases inhibition may induce the process of apoptosis. Finally, Mo-CBP 2 did not have a cytotoxic effect in mammalian Vero cells. This study highlights the biotechnological potential of Mo-CBP 2 as a promising molecule with low toxicity and potent activity. Further studies should be performed to better understand its mode of action and toxicity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: This study aims to evaluate the effective of azoles and MTX for patients with invasive candidiasis., Methods: We used the disk diffusion assay and the checkerboard assay to evaluate the in vitro interactions between MTX and antifungals. In addition, we used the transmission electron microscopy to observe the ultrastructure of the effect of MTX and fluconazole on Candida albicans., Results: The rates of synergy for the combination of MTX with fluconazole (FLC), itraconazole (ITC), and voriconazole (VRZ) were 91.3%, 65.2%, and 87% in checkerboard testing. No antagonism was found between methotrexate and azole antifungals in any of the strains. Furthermore, MTX treated C. albicans showed extensive cell wall vacuolations and the inhibition of blastospores growth, as observed using transmission electron microscopy. There was an apparent destruction of the cell membrane and cell wall resulting in the destruction of cytoplasm, a phenomenon observed when MTX was combined with azoles., Conclusion: This study provides evidence that the combination of azoles and MTX is effective for patients with invasive candidiasis, which on the other hand, will reduce the side effects of the drugs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

There is a growing clinical demand for topical itraconazole (ITC) delivery systems because of the expanding potential of the drug for topical fungal and non-fungal applications. Lipid-based nanocarriers offer great promise in this respect. In the present study, a new topical ITC gel based on lipid nanocapsules (LNC) was developed. ITC-LNC were compared to ITC-loaded nanostructured lipid carriers (ITC-NLC) with more established benefits as topical vectors. Both nanocarriers showed high entrapment efficiency (EE > 98%). Compared to ITC-NLC, the ITC-LNC showed a significantly smaller particle size (∼50 vs 155 nm), narrower size distribution (0.09 vs 0.38), faster initial release rate under sink conditions and greater in vitro antifungal activity against Candida albicans (C. albicans) (inhibition zone 29.4 vs 26.4 mm). ITC-LNC and ITC-NLC-based gels significantly enhanced the dermal retention of ITC in excised human skin relative to a conventional ITC gel. Histopathological assessment of a 14-day treatment of induced cutaneous candidiasis in a rat model indicated efficacy of the gel preparations. Fungal elements developed in the superficial epidermal skin layer were cleared by the end of treatment. Equally important, no histopathological changes in the epidermal and dermal layers of rat skin were observed. Findings of this study verified efficacy of topical ITC in the treatment of superficial fungal infections as well as effectiveness of LNC as biomimetic nanocarrier for dermal drug delivery. Combining ITC and LNC would present a bioactive nanocarrier system with good potentials for fungal infections and other skin applications., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Accurate prediction of cytochrome P450 (CYP) 3A activity in the early stage of drug development and in clinical practice is important. This study aimed to evaluate the previously constructed CYP3A activity prediction model after administration of CYP3A inhibitors and inducers and to modify the model for better prediction of CYP3A activity. Healthy male subjects received the following study drugs during three study periods: midazolam alone (control phase); midazolam with 200 mg of itraconazole (CYP3A inhibition phase); and midazolam with 150 mg of rifampicin (CYP3A induction phase). We quantified the concentrations of several endogenous CYP3A markers in both urine and plasma using gas chromatography-mass spectrometry. The urinary markers, including 6β-hydroxy (OH)-cortisol/cortisol, 6β-OH-cortisone/cortisone, 16α-OH-dehydroepiandrosterone (DHEA)/DHEA, 16α-OH-androstenedione (A-dione)/A-dione and 7β-OH-DHEA/DHEA, were significantly correlated with midazolam clearance in both the CYP3A inhibition and induction phases. We constructed a statistical prediction model after integrating data from a previous study to predict midazolam clearance as follows: Ln(midazolam clearance) = 2.5545 + 0.3988 × ln(7β-OH-DHEA/DHEA) + 0.1984 × ln(16α-OH-DHEA/DHEA) + 0.5031 × ln(6β-OH-cortisol/cortisol) - 0.1261 [ln(7β-OH-DHEA/DHEA) × ln(6β-OH-cortisol/cortisol)] (r 2  = 0.75). We suggest that quantitating endogenous markers in vivo coupled with the statistical prediction model may be useful for predicting CYP3A parameters., (Copyright © 2019 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

The emergence of drug-resistant fungal strains remains a severe threat for the public health, which prompts strict restrictions on the uses of antifungal drugs. However, the majority of lipophilic fungistatic agents are poorly water soluble with a low oral adsorption characteristic posing challenges for the precise prescriptions. In this study, a natural antimicrobial cationic peptide of epsilon-poly-l-lysine (EPL) decorated ordered mesoporous silica (SBA-15) was facilely prepared for the efficient loading of antifungal itraconazole (ITZ) drugs. The characterized mesoporous SBA-15/EPL/ITZ composite exhibited remarkable antifungal performance against Aspergillus fumigatus as a model mold, which was attributed to synergistic antifungal activities of ITZ and EPL in the mesopores. Moreover, the in vitro release behaviors of ITZ in the composite nanoexcipients both in simulated gastric fluid and fasted state simulated intestinal fluid were studied. The observed release kinetics of ITZ demonstrated a contributing role of SBA-15/EPL to enhance the solubility of ITZ and thereby may promote its flux across the gastrointestinal epithelium, which is beneficial for the absorption of drugs. Additionally, SBA-15/EPL/ITZ composites showed desirable biocompatibility toward mammalian red blood cells, human cervical cancer cells (Hela) and human embryonic kidney cells (HEK-293T). Furthermore, the pharmacokinetic profiles of obtained nano-formulations were assessed in rats, among which the improved adsorption of SBA-15/EPL/ITZ composites (AUC 0-24h sum : 8381.7 nM·h) was identified compared with that of pure ITZ (525.1 nM·h) and the commercial drug of Sporanox (7516.6 nM·h). Collectively, the prepared SBA-15/EPL/ITZ provides an ecofriendly and integrated nanocomposite with enhanced solubility of lipophilic drugs to combat proliferations of infectious fungi., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Itraconazole is an antagonist of the component Smoothened of Hedgehog pathway, which can inhibit the growth of medulloblastoma, basal cell carcinoma, and melanoma, etc. To research the binding mechanism of the Smoothened and triazoles, we used docking and molecular dynamics simulations on the Smoothened crystal structure and six triazoles. Unlike vismodegib, itraconazole can effectively bind into the pocket in the C-terminal domain of the Smoothened crystal structure instead of the N-terminal domain. The binding of itraconazole can change the conformation of the N-terminal domain even although itraconazole only had limited area contacting with N-terminal domain of the Smoothened. Besides, the binding of Itraconazole will not affect the binding of vismodegib. The strong binding affinity could be demonstrated between itraconazole and the Smoothened. Posaconazole and ketoconazole also had the strong binding affinity and the similar binding mode with the Smoothened crystal structure., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Dengue virus (DENV) is associated with an estimated 390 million infections per year, occurring across approximately 100 countries in tropical and sub-tropical regions. To date, there are no antiviral drugs or specific therapies to treat DENV infection. Posaconazole and itraconazole are potent antifungal drugs that inhibit ergosterol biosynthesis in fungal cells, but also target a number of human proteins. Here, we show that itraconazole and posaconazole have antiviral activity against DENV. Posaconazole inhibited replication of multiple serotypes of DENV and the related flavivirus Zika virus, and reduced viral RNA replication, but not translation of the viral genome. We used a combination of knockdown and drug sensitization assays to define the molecular target of posaconazole that mediates its antiviral activity. We found that knockdown of oxysterol-binding protein (OSBP) inhibited DENV replication. Moreover, knockdown of OSBP, but not other known targets of posaconazole, enhanced the inhibitory effect of posaconazole. Our findings imply OSBP as a potential target for the development of antiviral compounds against DENV., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Itraconazole (ITZ) is a well-known, FDA-approved antifungal drug that is also in clinical trials for its anticancer activity. ITZ exerts its anticancer activity through several disparate targets and pathways. ITZ inhibits angiogenesis by hampering the functioning of the vascular endothelial growth receptor 2 (VEGFR2) and by indirectly inhibiting mTOR signaling. Furthermore, ITZ directly inhibits the growth of several types of tumor cells by antagonizing Hedgehog signaling. Recently, we reported that ITZ also has broad-spectrum antiviral activity against enteroviruses, cardioviruses and hepatitis C virus, independent of established ITZ-activities but instead via a novel target, oxysterol-binding protein (OSBP), a cellular lipid shuttling protein. In this study, we analyzed which structural features of ITZ are important for the OSBP-mediated antiviral activity. The backbone structure, consisting of five rings, and the sec-butyl chain are important for antiviral activity, whereas the triazole moiety, which is critical for antifungal activity, is not. The features required for OSBP-mediated antiviral activity of ITZ overlap mostly with published features required for inhibition of VEGFR2 trafficking, but not Hh signaling. Furthermore, we use in silico studies to explore how ITZ could bind to OSBP. Our data show that several pharmacological activities of ITZ can be uncoupled, which is a critical step in the development of ITZ-based antiviral compounds with greater specificity and reduced off-target effects., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

In this work, we aimed at developing an improved topical SLN formulation combining itraconazole delivery with a coating layer of didodecyldimethylammonium bromide, thus repurposing the drug effectiveness by synergistic skin anticancer effectiveness. In order to obtain a stable SLN formulation with small homogeneously dispersed particles, a deep formulative study was developed screening three different solid lipids (Suppocire NB, Cetyl Palmitate and Dynasan 114) for the SLN preparation by the phase inversion temperature. A bluishcolored shade formulation, with homogeneous small particles size (<50 nm) was obtained only using Suppocire NB. The cytotoxicity of all SLN was tested after 24 h exposure against three adherent skin cell lines (A431, HaCaT and SK-MEL-5). Results demonstrate that both unloaded and drugloaded SLN did not significantly affect the cell viability of the non-tumoral HaCaT cell line, thus confirming the safe potential topical application of these formulations. A dose-dependent decrease in cell viability was observed for the tumoral cell lines, A431 and SK-MEL-5, with a significant reduction of the A431 cancer cell line viability. The drug molecule addition to the uncoated nanoparticles was able to increase of almost 20% the reduction of the viability of the cancer cells treated. Ours results demonstrate the potentiality of repurposing itraconazole activity by using the combined nanoencapsulation strategy with the positively charged coating layer on SLN, which can be further investigated as a promising stable and safe approach to significantly reduce the viability of skin cancer cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The European Society for Clinical Microbiology and Infectious Diseases, the European Confederation of Medical Mycology and the European Respiratory Society Joint Clinical Guidelines focus on diagnosis and management of aspergillosis. Of the numerous recommendations, a few are summarized here. Chest computed tomography as well as bronchoscopy with bronchoalveolar lavage (BAL) in patients with suspicion of pulmonary invasive aspergillosis (IA) are strongly recommended. For diagnosis, direct microscopy, preferably using optical brighteners, histopathology and culture are strongly recommended. Serum and BAL galactomannan measures are recommended as markers for the diagnosis of IA. PCR should be considered in conjunction with other diagnostic tests. Pathogen identification to species complex level is strongly recommended for all clinically relevant Aspergillus isolates; antifungal susceptibility testing should be performed in patients with invasive disease in regions with resistance found in contemporary surveillance programmes. Isavuconazole and voriconazole are the preferred agents for first-line treatment of pulmonary IA, whereas liposomal amphotericin B is moderately supported. Combinations of antifungals as primary treatment options are not recommended. Therapeutic drug monitoring is strongly recommended for patients receiving posaconazole suspension or any form of voriconazole for IA treatment, and in refractory disease, where a personalized approach considering reversal of predisposing factors, switching drug class and surgical intervention is also strongly recommended. Primary prophylaxis with posaconazole is strongly recommended in patients with acute myelogenous leukaemia or myelodysplastic syndrome receiving induction chemotherapy. Secondary prophylaxis is strongly recommended in high-risk patients. We strongly recommend treatment duration based on clinical improvement, degree of immunosuppression and response on imaging., (Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Parechovirus A3 (Par-A3, formerly human parechovirus 3) is an emerging viral infection of the central nervous system in children. We used an automated, homogeneous, cell based assay to identify itraconazole and posaconazole as inhibitors of Par-A3, with antiviral activity below concentrations clinically attainable in pediatric patients. Currently, there is no approved antiviral treatment for Par-A3 infection, despite numerous reports of serious Par-A3 disease in neonates and infants., (Copyright © 2017. Published by Elsevier B.V.)

Sporotrichosis is an emerging chronic, granulomatous, subcutaneous, mycotic infection caused by Sporothrix species. Sporotrichosis is treated with the azole drug itraconazole as ketoconazole is ineffective. It is a well-known fact that azole drugs act by inhibiting cytochrome P450 monooxygenases (P450s), heme-thiolate proteins. To date, nothing is known about P450s in Sporothrix schenckii and the molecular basis of its resistance to ketoconazole. Here we present genome-wide identification, annotation, phylogenetic analysis and comprehensive P450 family-level comparative analysis of S. schenckii P450s with pathogenic fungi P450s, along with a rationale for ketoconazole resistance by S. schenckii based on in silico structural analysis of CYP51. Genome data-mining of S. schenckii revealed 40 P450s in its genome that can be grouped into 32 P450 families and 39 P450 subfamilies. Comprehensive comparative analysis of P450s revealed that S. schenckii shares 11 P450 families with plant pathogenic fungi and has three unique P450 families: CYP5077, CYP5386 and CYP5696 (novel family). Among P450s, CYP51, the main target of azole drugs was also found in S. schenckii. 3D modeling of S. schenckii CYP51 revealed the presence of characteristic P450 motifs with exceptionally large reductase interaction site 2. In silico analysis revealed number of mutations that can be associated with ketoconazole resistance, especially at the channel entrance to the active site. One of possible reason for better stabilization of itraconazole, compared to ketoconazole, is that the more extended molecule of itraconazole may form a hydrogen bond with ASN-230. This in turn may explain its effectiveness against S. schenckii vis-a-vis resistant to ketoconazole. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Triazole antimycotic itraconazole contains in its structure three chiral centres; therefore, it forms eight stereoisomers. Commercial preparations of itraconazole are a mixture of four cis-diastereoisomers. There is much evidence that efficacy, adverse effects, and toxicity of chiral drugs may be stereospecific. Therefore, we have prepared 4 pure cis-diastereoisomers of itraconazole and investigated their effects on transcriptional activities of xenoreceptors aryl hydrocarbon receptor AhR and pregnane X receptor PXR. Gene reporter assays showed that itraconazole dose-dependently activated both AhR and PXR, and the activation of AhR but not of PXR was enantiospecific. Itraconazole diastereoisomers transformed AhR and PXR into their DNA-binding forms, as demonstrated by electromobility shift assays. Cytochrome P450 CYP1A1 mRNA and protein were induced by itraconazole diastereoisomers in human hepatoma cells HepG2, human skin cells HaCaT, and in primary human hepatocytes. The expression of CYP3A4 in human intestinal LS180 cells was not influenced by itraconazole, but we observed downregulation of CYP3A4 in human hepatocytes. Collectively, we show that itraconazole is a dual activator of AhR and PXR, with differential effects on the target genes for xenoreceptors. The enantiospecific pattern was observed only in gene reporter assays for AhR. The data presented here might be of toxicological and clinical importance., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Introduction: The importance of drug-induced effects on the inotropic state of the heart is well known. Unlike hemodynamic and cardiac electrophysiological methods, which have been routinely used in drug safety testing for years, the non-clinical assessment of drug effects on myocardial contractility is used less frequently with no established translation to humans. The goal of these studies was to determine whether assessment of alternate measures of cardiac inotropy could detect drug-induced changes in the contractile state of the heart using drugs known to have clinically relevant positive and negative effects on myocardial contractility. This study also evaluated drug-induced effects on lusitropy (relaxation) parameters of the heart., Methods: A double 4×4 Latin square study design using Beagle dogs (n=8) was conducted. Drugs were administrated orally. Arterial blood pressure (BP), left ventricular pressure (LVP) and the electrocardiogram (ECG) were assessed across different laboratories using the same protocol. Each of the six laboratories studied at least 2 drugs (one positive inotrope (pimobendan or amrinone) and one negative inotrope (itraconazole or atenolol) at 3 doses selected to match clinical exposure data and a vehicle control). Animals were instrumented with an ITS telemetry system or DSI's D70-PCTP or PhysioTel™ Digital system. The data acquisition and analysis systems used were Ponemah, Notocord or EMKA., Results: The derived inotropic and lusitropic parameters evaluated included peak systolic and end diastolic LVP, LVdP/dt max , LVdP/dt 40 , QA interval, LVdP/dt min and Tau. This study showed that LVdP/dt 40 provided essentially identical results to LVdP/dt max qualifying it as an index to assess drug effects on cardiac contractility. LVdP/dt 40 provided an essentially identical assessment to that of LVdP/dt max . The QA interval did not react sensitively to the drugs tested in this study; however, it did detect large effects and could be useful in early cardiovascular safety studies. The lusitropic parameter, LVdP/dt min , was modestly decreased, and Tau was increased, by atenolol and itraconazole. At the doses tested, amrinone and pimobendan produced no changes in LVdP/dt min while Tau was modestly increased. The drugs did not produce effects on BP, HR or the ECG at the doses tested. Blood samples were drawn to confirm drug exposures predicted from independent pharmacokinetic studies., Discussion: These findings indicate that this experimental model can accurately and consistently detect changes in cardiac contractility, across multiple sites and instrumentation systems. While LVdP/dt 40 produced responses similar to LVdP/dt max , the QA interval and lusitropic parameters LVdP/dt min and Tau were not markedly changed at the dose of drugs tested. Further studies with drugs that affect early diastolic relaxation through calcium handling are needed to better evaluate drug-induced changes on lusitropic properties of the heart., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The genus Enterovirus (e.g. poliovirus, coxsackievirus, rhinovirus) of the Picornaviridae family of positive-strand RNA viruses includes many important pathogens linked to a range of acute and chronic diseases for which no approved antiviral therapy is available. Targeting a step in the life cycle that is highly conserved provides an attractive strategy for developing broad-range inhibitors of enterovirus infection. A step that is currently explored as a target for the development of antivirals is the formation of replication organelles, which support replication of the viral genome. To build replication organelles, enteroviruses rewire cellular machinery and hijack lipid homeostasis pathways. For example, enteroviruses exploit the PI4KIIIβ-PI4P-OSBP pathway to direct cholesterol to replication organelles. Here, we uncover that TTP-8307, a known enterovirus replication inhibitor, acts through the PI4KIIIβ-PI4P-OSBP pathway by directly inhibiting OSBP activity. However, despite a shared mechanism of TTP-8307 with established OSBP inhibitors (itraconazole and OSW-1), we identify a number of notable differences between these compounds. The antiviral activity of TTP-8307 extends to other viruses that require OSBP, namely the picornavirus encephalomyocarditis virus and the flavivirus hepatitis C virus., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Azole antifungals are used not only clinically for fungal infections but also used as agricultural fungicides. Recently, azole-resistant Aspergillus fumigatus containing a tandem repeat in the promoter region of cyp51A combined with amino acid substitution(s) appears in the environment in Eurasia, especially in several European countries. Although azole fungicides have been used in Japan, especially in Hokkaido, surveillance and characterization of A. fumigatus in Hokkaido have not been reported. In this study, we collected soil samples from farms that used an azole fungicide in the Tokachi area of eastern Hokkaido, isolated 91 A. fumigatus strains, and determined the minimal inhibitory concentrations of medical azoles required for these strains. Moreover, because causative agent A. fumigatus is ubiquitous in the air and acquired from the environment, we collected 22 clinical isolates of A. fumigatus to measure their susceptibility to medical azoles in a hospital in the Tokachi area. Our data show that almost all A. fumigatus isolates retained susceptibility to medical azoles. Clinical isolates OKH34 and OKH6 showed 8 and 2 μg/mL of voriconazole, respectively, as the minimal inhibitory concentration. Both isolates did not contain tandem repeat in cyp51A promoter region. An isolate contained G448S mutation in cyp51A conferring voriconazole resistance, which is the first report from Japan. Our data shows the existence of azole-resistant and low azole-susceptible clinical isolates and highlight the necessity for continuous surveillance in Japan because resistant A. fumigatus strains can arise through clinical or environmental selection or could be introduced from overseas., (Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Itraconazole (ITZ)-loaded microemulsion (ME) systems for intranasal (IN) delivery were developed for the treatment of human rhinovirus serotype 1B (HRV1B) infection. ITZ was incorporated into the oil-in-water (o/w) ME formulation composed of benzyl alcohol (oil), Cremophor EL (surfactant), Solutol HS15 (cosurfactant), and water. The optimized composition of ME was determined by constructing pseudo-ternary phase diagram. ITZ ME formulation with about 150nm mean diameter and spherical shape was prepared and the solubility of ITZ in blank ME was markedly improved (up to 13.9mg/mL). The initial value of droplet size was maintained with four times dilution in the aqueous buffer and 72h incubation. Released amounts of drug from ME formulation were significantly enhanced compared to drug suspension group (p<0.05). Particularly, ITZ ME group displayed lower levels of inflammatory markers in the lung compared to ITZ suspension group after their IN administration in the HRV1B-infected mouse model (p<0.05). Developed ITZ ME formulation via IN route can be a promising candidate for the treatment of rhinovirus infection., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Aims: The incidence of secondary systemic fungal infections has sharply increased in bacterial septic patients. Antimycotics exhibit immunomodulatory properties, yet these effects are incompletely understood in secondary systemic fungal infections following bacterial sepsis. We investigated a model of systemic inflammation to determine whether antimycotics (liposomal amphotericin B (L-AMB), itraconazol (ITC), and anidulafungin (ANI)) modulate the gene and protein expression as well as the phagocytic activity of lipopolysaccharide (LPS)-stimulated human monocytes., Main Methods: THP-1 monocytes were incubated with L-AMB, ITC or ANI and LPS. Gene expression levels of cytokines (TNF-, IL-1, IL-6, and IL-10) were measured after 2h, 6h, and 24h. Cytokine protein levels were evaluated after 24h and phagocytic activity was determined following co-incubation with Escherichia coli., Key Findings: All antimycotics differentially modulated the gene and protein expression of cytokines in sepsis-like conditions. In the presence of LPS, we identified L-AMB as immunosuppressive, whereas ITC demonstrated pro-inflammatory properties. Both compounds induced remarkably less phagocytosis., Significance: Our study suggests that antimycotics routinely used in septic patients alter the immune response in sepsis-like conditions by modulating cytokine gene and protein expression levels and phagocytic activity. Future treatment strategies should consider the immune status of the host and apply antimycotics accordingly in bacterial septic patients with secondary fungal infections., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Various antifungal agents both topical and systemic have been introduced into clinical practice for effectively treating dermatophytic conditions. Dermatophytosis is the infection of keratinised tissues caused by fungal species of genera Trichophyton, Epidermophyton and Microsporum, commonly known as dermatophytes affecting 20-25% of the world's population. The present study aims at determining the susceptibility patterns of dermatophyte species recovered from superficial mycoses of human patients in Himachal Pradesh to antifungal agents; itraconazole, terbinafine and ketoconazole. The study also aims at determining the minimum inhibitory concentrations (MICs) of these agents following the recommended protocol of Clinical and Laboratory Standards Institute (CLSI) (M38-A2)., Methodology: A total of 53 isolates of dermatophytes (T. mentagrophyte-34 in no., T. rubrum-18 and M. gypseum-1) recovered from the superficial mycoses were examined. Broth microdilution method M38-A2 approved protocol of CLSI (2008) for filamentous fungi was followed for determining the susceptibility of dermatophyte species., Results: T. mentagrophyte isolates were found more susceptible to both itraconazole and ketoconazole as compared to terbinafine (MIC50: 0.125 µg/ml for itraconazole, 0.0625 µg/ml for ketoconazole and 0.5 µg/ml for terbinafine). Three isolates of T. mentagrophytes (VBS-5, VBSo-3 and VBSo-73) and one isolate of T. rubrum (VBPo-9) had higher MIC values of itraconazole (1 µg/ml). Similarly, the higher MIC values of ketoconazole were observed in case of only three isolates of T. mentagrophyte (VBSo-30 = 2 µg/ml; VBSo-44, VBM-2 = 1 µg/ml). The comparative analysis of the three antifungal drugs based on t-test revealed that 'itraconazole and terbinafine' and 'terbinafine and ketoconazole' were found independent based on the P < 0.005 in case of T. mentagrophyte isolates. In case of T. rubrum, the similarity existed between MIC values of 'itraconazole and ketoconazole' and 'terbinafine and ketoconazole'., Conclusion: The MIC values observed in the present study based on standard protocol M38-A2 of CLSI 2008 might serve as reference for further studies covering large number of isolates from different geographic regions of the state. Such studies might reflect on the acquisition of drug resistance among isolates of dermatophyte species based on MIC values.

Introduction: Drug-induced effects on the cardiovascular system remain a major cause of drug attrition. While hemodynamic (blood pressure (BP) and heart rate (HR)) and electrophysiological methods have been used in testing drug safety for years, animal models for assessing myocardial contractility are used less frequently and their translation to humans has not been established. The goal of these studies was to determine whether assessment of contractility and hemodynamics, when measured across different laboratories using the same protocol, could consistently detect drug-induced changes in the inotropic state of the heart using drugs known to have clinically relevant positive and negative effects on myocardial contractility., Methods: A 4×4 double Latin square design (n=8) design using Beagle dogs was developed. Drugs were administrated orally. Arterial blood pressure, left ventricular pressure (LVP) and the electrocardiogram were assessed. Each of the six laboratories studied at least 2 drugs (one positive inotrope (pimobendan or amrinone) and one negative inotrope) (itraconazole or atenolol) at 3 doses selected to match clinical exposure data and a vehicle control. Animals were instrumented with an ITS telemetry system, DSI's D70-PCTP system or DSI's Physiotel Digital system. Data acquisition and analysis systems were Ponemah, Notocord or EMKA., Results: Derived parameters included: diastolic, systolic and mean arterial BP, peak systolic LVP, HR, end-diastolic LVP, and LVdP/dtmax as the primary contractility index. Blood samples were drawn to confirm drug exposures predicted from independent pharmacokinetic studies. Across the laboratories, a consistent change in LVdP/dtmax was captured despite some differences in the absolute values of some of the hemodynamic parameters prior to treatment., Discussion: These findings indicate that this experimental model, using the chronically instrumented conscious dog, can accurately and consistently detect changes in cardiac contractility, across multiple sites and instrumentation systems, and that data obtained in this model may also translate to clinical outcomes., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Aspergillus fumigatus is the most important etiological agent of invasive aspergillosis. Recently, an increasing number of azole-resistant A. fumigatus isolates have been described in various countries. The prevalence of azole resistance was investigated in this study using our culture collection of A. fumigatus isolates collected between 1999 and 2012 from clinical specimens. Seven hundred and forty-six A. fumigatus isolates, collected from 419 patients, were investigated. First, all isolates were screened for resistance to itraconazole by subculturing on Sabouraud dextrose agar that contained 4 mg/L itraconazole. For isolates that grew on the itraconazole containing agar, the in vitro activities of amphotericin B, itraconazole, voriconazole and posaconazole were determined using the Clinical and Laboratory Standards Institute (CLSI) M38-A reference method. After PCR amplification, the full sequence of the cyp51A gene and its promoter region was determined for all in vitro azole-resistant isolates. Itraconazole resistance was found in 10.2% of the A. fumigatus isolates. From 2000 onwards, patients were observed annually with an itraconazole-resistant isolate. According to in vitro susceptibility tests, amphotericin B exhibited good activity against all isolates whereas the azoles were resistant. Sequence analysis of the promoter region and CYP51A gene indicated the presence of TR34/L98H in 86.8% (n = 66) of isolates. This initial analysis of the resistance mechanism of A. fumigatus from Turkey revealed a common TR34/L98H mutation in the cyp51A gene., (Copyright © 2015. Published by Elsevier Ltd.)

The incidence of Aspergillus infection has been increasing in the past few years. Also, new Aspergillus fumigatus-related species, namely Aspergillus lentulus, Aspergillus udagawae, and Aspergillus viridinutans, were shown to infect humans. These fungi exhibit marked morphological similarities to A. fumigatus, albeit with different clinical courses and antifungal drug susceptibilities. The present study used liquid chromatography/time-of-flight mass spectrometry to identify the secondary metabolites secreted as virulence factors by these Aspergillus species and compared their antifungal susceptibility. The metabolite profiles varied widely among A. fumigatus, A. lentulus, A. udagawae, and A. viridinutans, producing 27, 13, 8, and 11 substances, respectively. Among the mycotoxins, fumifungin, fumiquinazoline A/B and D, fumitremorgin B, gliotoxin, sphingofungins, pseurotins, and verruculogen were only found in A. fumigatus, whereas auranthine was only found in A. lentulus. The amount of gliotoxin, one of the most abundant mycotoxins in A. fumigatus, was negligible in these related species. In addition, they had decreased susceptibility to antifungal agents such as itraconazole and voriconazole, even though metabolites that were shared in the isolates showing higher minimum inhibitory concentrations than epidemiological cutoff values were not detected. These strikingly different secondary metabolite profiles may lead to the development of more discriminative identification protocols for such closely related Aspergillus species as well as improved treatment outcomes., (Copyright © 2015 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: The purpose of this study was to determine the species distribution and to monitor the antifungal susceptibility profiles of clinical Candida isolates collected in China from 2009 to 2013., Methods: The antifungal susceptibilities of 952 Candida isolates were tested., Results: Candida albicans was the most common species, accounting for 65.7% of the total isolates. The most frequently isolated non-albicans Candida species in this study was Candida glabrata (193, 20.3%). Nearly 7.6%, 3.2%, 1.8%, and 1.1% of the 952 isolates exhibited decreased susceptibility to fluconazole, voriconazole, itraconazole, and flucytosine, respectively. Moreover, seven C. albicans and one Candida krusei had an amphotericin B minimum inhibitory concentration (MIC) of 2 μg/ml., Conclusions: The distribution of species and the prevalence of antifungal resistance in Candida isolates varied among different areas in China. Continuous monitoring of resistance patterns is necessary to control the spread of resistance in clinical isolates of Candida species., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Ergosterol biosynthesis and homeostasis in the parasitic protozoan Trypanosoma brucei was analyzed by RNAi silencing and inhibition of sterol C24β-methyltransferase (TbSMT) and sterol 14α-demethylase [TbSDM (TbCYP51)] to explore the functions of sterols in T. brucei growth. Inhibition of the amount or activity of these enzymes depletes ergosterol from cells at <6 fg/cell for procyclic form (PCF) cells or <0.01 fg/cell for bloodstream form (BSF) cells and reduces infectivity in a mouse model of infection. Silencing of TbSMT expression by RNAi in PCF or BSF in combination with 25-azalanosterol (AZA) inhibited parasite growth and this inhibition was restored completely by adding synergistic cholesterol (7.8 μM from lipid-depleted media) with small amounts of ergosterol (1.2 μM) to the medium. These observations are consistent with the proposed requirement for ergosterol as a signaling factor to spark cell proliferation while imported cholesterol or the endogenously formed cholesta-5,7,24-trienol act as bulk membrane components. To test the potential chemotherapeutic importance of disrupting ergosterol biosynthesis using pairs of mechanism-based inhibitors that block two enzymes in the post-squalene segment, parasites were treated with AZA and itraconazole at 1 μM each (ED50 values) resulting in parasite death. Taken together, our results demonstrate that the ergosterol pathway is a prime drug target for intervention in T. brucei infection., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)

This study reports the use of variants of coherent anti-Stokes Raman scattering (CARS) microscopy as a novel method for improved physicochemical characterization of drug-loaded silica particles. Ordered mesoporous silica is a biomaterial that can be loaded to carry a number of biochemicals, including poorly water-soluble drugs, by allowing the incorporation of drug into nanometer-sized pores. In this work, the loading of two poorly water-soluble model drugs, itraconazole and griseofulvin, in MCM-41 silica microparticles is characterized qualitatively, using the novel approach of CARS microscopy, which has advantages over other analytical approaches used to date and is non-destructive, rapid, label free, confocal and has chemical and physical specificity. The study investigated the effect of two solvent-based loading methods, namely immersion and rotary evaporation, and microparticle size on the three-dimensional (3-D) distribution of the two loaded drugs. Additionally, hyperspectral CARS microscopy was used to confirm the amorphous nature of the loaded drugs. Z-stacked CARS microscopy suggested that the drug, but not the loading method or particle size range, affected 3-D drug distribution. Hyperspectral CARS confirmed that the drug loaded in the MCM-41 silica microparticles was in an amorphous form. The results show that CARS microscopy and hyperspectral CARS microscopy can be used to provide further insights into the structural nature of loaded mesoporous silica microparticles as biomaterials., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The study aimed to develop novel ITZ-loaded deformable liposomes (DL) in presence of hydroxypropyl-β-cyclodextrin (HPβCD) (DL-CD) to enhance antifungal activity. These formulations have been reported as conceivable vesicles to deliver drug molecules to the skin layers. The efficiency of the prepared systems was compared with conventional liposomes (CL) and ITZ solution. The developed liposomes were characterized for particle size, entrapment efficiency (EE %), deformability, stability, and morphology of the vesicles. In addition, ex vivo penetration and antifungal activity were evaluated. It was found that the presence of HPβCD played a significant role in reducing the vesicle size to nano range. The deformability study and TEM images revealed that membrane deformability of DL and DL-CD was much higher than that of CL. Moreover, DL-CD enhanced the amount of ITZ in SC and deeper skin layers compared to DL and CL. The antifungal activity of ITZ-loaded deformable liposomes remained intact compared to ITZ solution. It can be concluded that deformable liposomes in the presence of HPβCD may be a promising carrier for effective cutaneous delivery of ITZ., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Itraconazole (ITR) is commonly used in the treatment of Candida infections. It has a nephrotoxic effect and low bioavailability in patients who suffer from renal insufficiency, and its poor solubility in water makes ITR largely unavailable. Cyclodextrins (CyDs) are used to form inclusion complexes with drugs to improve their aqueous solubility and to reduce their side effects. In this study, ITR was complexed with γ-cyclodextrin (γ-CyD), hydroxypropyl-β-cyclodextrin (HP-β-CyD), methyl-β-cyclodextrin (Met-β-CyD) and sulphobutyl ether-β-cyclodextrin (SBE7-β-CyD) to increase its water solubility and to reduce the side effects of the drug without decreasing antifungal activity. Complex formation between ITR and CyDs was evaluated using SEM, (1)H NMR and XRD studies. The antifungal activity of the complexes was analyzed on Candida albicans strains, and the susceptibility of the strains was found to be higher for the ITR-SBE7-β-CyD complex than for the complexes that were prepared with other CyDs. Vaginal bioadhesive sustained release tablet formulations were developed using the ITR-SBE7-β-CyD inclusion complex to increase the residence time of ITR in the vagina, thereby boosting the efficacy of the treatment. The swelling, matrix erosion and bioadhesion properties of formulations and the drug release rate of these tablets were analyzed, and the most therapeutically effective vaginal formulation was determined., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Azole resistance in the fungal pathogen Aspergillus fumigatus is an emerging problem and may develop during azole therapy in humans and animals or exposure to azole fungicides in the environment. To assess the potential risk of azole-resistance emergence in avian farms where azole compounds are used for the control of avian mycoses, we conducted a drug susceptibility study including A. fumigatus isolates from birds and avian farms in France and Southern China. A total number of 175 isolates were analyzed: 57 isolates were collected in France in avian farms where chemoprophylaxis with parconazole was performed; 51 isolates were collected in southern China in avian farms where no chemoprophylaxis was performed; and 67 additional isolates came from the collection of a mycology laboratory. No resistant isolate was detected, and the distribution of minimum inhibitory concentrations was similar for isolates collected in farms with or without azole chemoprophylaxis. For 61 randomly selected isolates, the full coding sequence of the Cyp51A gene was determined to detect mutations. Nine amino acid alterations were found in the target enzyme, 3 of which were new.

A variety of studies indicate that itraconazole possesses greater intrinsic activity compared to the other azole derivatives against Candida parapsilosis. Efficacy has never been tested in an experimental setting. To this end, C. parapsilosis was used for challenge of 117 rats rendered neutropenic after a course of cyclophosphamide. Rats were assigned to receive intravenous treatment with saline (group A); itraconazole q12h (group B); fluconazole q12h (group C); single dose of ceftriaxone and saline (group D); single dose of ceftriaxone and itraconazole q12h (group E); and single dose of ceftriaxone and fluconazole q12h (group F). Survival was recorded, and yeast outgrowth of liver, spleen, lung, and kidney was measured after sacrifice at serial time intervals. Growth of the test isolate in tissues was significantly lower in group B than in groups A and C after 72 h. However, outgrowth of enterobacteria was found in tissues of groups A, B, and C, implying a phenomenon of bacterial translocation from the gut. When this phenomenon was suppressed with single doses of ceftriaxone, a striking survival benefit of itraconazole-treated animals was found (p = 0.022, group E vs. group F). The present results suggest than in deep infections by C. parapsilosis intravenously administered intraconazole may eradicate the offending agent and provide survival benefit when chemotherapy-induced bacterial translocation from the gut is suppressed. Further clinical evidence is required to support these findings.

The clinicopathological and microbiological features of chromoblastomycosis caused by Fonsecaea pedrosoi or Fonsecaea monophora are summarized. Four F. monophora and three F. pedrosoi strains were isolated from seven consecutive chromoblastomycosis patients and identified by genetic analysis between 2004 and 2012 in a teaching hospital in southern China. Six strains were sensitive to voriconazole, itraconazole and terbinafine using E-test and Neo-Sensitabs. Six patients healed after oral itraconazole or terbinafine, and one was lost to follow up. Internal transcribed spacer sequence is sufficient for species delimitation of Fonsecaea, and the Neo-Sensitabs test and E-test are comparable in their susceptibility testing. Itraconazole and/or terbinafine may be the preferred treatment for this chromoblastomycosis., (© 2012 The Authors Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.)