Voriconazole Research Articles

Study on the Inhibitory Activity and Possible Mechanism of Myriocin on Clinically Relevant Drug-Resistant Candida albicans and Its Biofilms.

In order to prevent and control the infection of Candida albicans, the antifungal activity, possible mechanism of myriocin against C. albicans and its biofilm were studied. The antifungal activity of myriocin was investigated by microdilution method. The effect of myriocin on fungal cell wall or membrane was evaluated by adding sorbitol, ergosterol or phytosphingosine (PHS). The damage to the cell membrane was investigated with propidium iodide (PI) staining and visualized by scanning electron microscope (SEM). The effects on biofilms and extracellular polysaccharides (EPS) were observed by crystal violet staining method and phenol-sulfuric acid method respectively. The adhesion of C. albicans cells to hydrocarbons was tested to evaluate cell surface hydrophobic (CSH). The combined effects of myriocin and antifungal drugs commonly used in clinical practice were investigated by using the checkerboard microdilution method. Minimal inhibitory concentrations (MICs) were found to be 0.125-4 µg/mL. Myriocin was found to affect both cell wall and cell membrane. After exposure to myriocin, biofilm and EPS were found to be inhibited and removed, and the CSH was decreased. The combined fungistasis of myriocin and voriconazole (VCZ) or amphotericin B (AMB) were additive. Myriocin had significant antifungal activity against C. albicans, and the antifungal mechanisms might be cell wall and membrane damage. Myriocin effectively inhibited and eliminated biofilms, and its mechanism may be related to the inhibition of EPS and CSH.

For Treatment of Invasive Aspergillosis, Posaconazole Was Noninferior to Voriconazole

Although voriconazole is recommended as a first-line treatment for patients with invasive pulmonary aspergillosis, its adverse event profile has

Azole Resistance in Aspergillus fumigatus: A Five-Year Follow Up Experience in a Tertiary Hospital With a Special Focus on Cystic Fibrosis.

Azole-resistant Aspergillus fumigatus (ARAf) has emerged worldwide during the last decades. Drug pressure after long term treatments of chronically infected patients and the propagation of environmental clones selected under the pressure of imidazoles fungicides used in agriculture and farming both account for this emergence. The objectives of this study were to determine the rate of azole resistance in Aspergillus fumigatus during a 5-year period, taking into account (i) differences between underlying diseases of the patients treated, (ii) cross-resistance between azoles, and (iii) focusing on the 5-year evolution of our center’s cystic fibrosis cohort. Overall, the rates of voriconazole (VRC)-resistant and itraconazole (ITC)-resistant A. fumigatus isolates were 4.1% (38/927) and 14.5% (95/656), respectively, corresponding to 21/426 (4.9%) and 44/308 (14.3%) patients, respectively. Regarding cross-resistance, among VRC-R isolates tested for ITC, nearly all were R (20/21;95%), compared to only 27% (20/74) of VRC-R among ITC-R isolates. The level of azole resistance remained somewhat stable over years but greatly varied according to the azole drug, patient origin, and clinical setting. Whereas azole resistance during invasive aspergillosis was very scarce, patients with cystic fibrosis were infected with multiple strains and presented the highest rate of resistance: 5% (27/539) isolates were VRC-R and 17.9% (78/436) were ITC-R. These results underline that the interpretation of the azole resistance level in Aspergilllus fumigatus in a routine setting may consider the huge variability depending on the azole drug, the clinical setting, the patient background and the type of infection.

CYP51A1 polymorphism and voriconazole-associated hepatotoxicity in children undergoing hematopoietic cell transplant.

Fungal CYP51A (14α-sterol demethylase) is the target of an azole antifungal, voriconazole (VCZ), which also partially inhibits human CYP51A1. Hepatotoxicity is a common adverse effect of azoles, which is reported to be caused by altered gene expressions secondary to cholesterol synthesis inhibition by azoles. This is a post-hoc analysis of a previously conducted phase 1 dose-finding study of prophylactic VCZ in 56 pediatric hematopoietic cell transplant recipients. We explored an association between variants in human CYP51A1 (rs2282976 and rs6465348) and VCZ-induced hepatotoxicity. Genotype A/G or G/G in rs6465348 showed lower odds of hepatotoxicity after adjusting for VCZ area-under-the-curve (OR: 0.10, 95% CI: 0.01 – 0.79, vs. A/A).

Reversal of azole resistance in Candida albicans by oridonin

ObjectivesCandida albicans is a yeast that causes fungal infections with high mortality and is typically resistant to azole drugs. To overcome this resistance, we explored the combined use of oridonin (ORI) and three azole drugs, namely fluconazole (FLC), itraconazole (ITR) and voriconazole (VOR). Azole-resistant C. albicans strains were obtained from cancer patients and the reversal of drug resistance in these strains was investigated. MethodsThe synergistic antifungal activity of ORI and azole drugs was measured by checkerboard microdilution and time–kill assays. The resistance reversal mechanisms, namely inhibition of drug efflux and induction of apoptosis, were investigated by flow cytometry. Expression levels of the efflux pump-related genesCDR1 and CDR2 were assessed by RT-qPCR. ResultsThe efflux pump inhibition assay with ORI showed that the minimum inhibitory concentrations (MICs) of FLC (128-fold), ITR (64-fold) and VOR (250-fold) decreased significantly. Upregulation of genes encodingCDR1 and CDR2 was confirmed in the resistant strain. The sensitising effect of ORI on FLC in the treatment of C. albicans also included the promotion of apoptosis. ConclusionWe demonstrated that combining azoles with ORI exerted potent synergism and that ORI could promote sensitisation to azoles in azole-resistantC. albicans. The discovery that ORI can effectively inhibit drug efflux and promote apoptosis may provide new insights and therapeutic strategies to overcome increasing azole resistance in C. albicans.

Application of the Westgard Multi-rule Theory to Internal Quality Control Evaluation of Voriconazole for Therapeutic Drug Monitoring

Background: There is no worldwide recognized reference internal quality control method for Therapeutic Drug Monitoring (TDM) of voriconazole (VCZ) by Liquid Chromatography (LC). In this study, we aimed to develop an internal quality control method for TDM of VCZ, evaluate it by the Westgard multi-rule theory, and guarantee the analytical quality of the assays. Methods: The plasma concentration of VCZ was detected by two-dimensional liquid chromatography with ultraviolet detection (2D-LC-UV) method. The internal quality control results accompanying with TDM of VCZ in our laboratory from July 2019 to January 2020 were collected and retrospectively studied. The Levey-Jennings quality chart and Z-score quality chart were drawn and Westgard multirules of 12s/13s/22s/R4s/41s/10x were applied to assess the suitable quality control method for TDM of VCZ. Results: The 2D-LC-UV method was well suited to monitor the plasma concentration of VCZ and increase the real-time capability of TDM for VCZ. Combined with Westgard multi-rules, the quality control charts of Levery-Jennings and Z-score both can timely discover and judge the systematic errors and random errors for the internal quality control results. 86 batches of quality control products were assessed and 7 times warnings and 6 times out of control were detected. Conclusion: The Westgard multi-rules, with high efficacy in determining detection errors, has important application value in the internal quality control for TDM of VCZ. The developed quality control method can improve the accuracy and reliability for VCZ measurement by the 2D-LC-UV method and further promote the clinical rational use of the drug.

Correlation between enzyme multiplied immunoassay technique and high-performance liquid chromatography in the quantification of voriconazole in a paediatric population

The enzyme multiplied immunoassay technique (EMIT) is a new method for determining the plasma concentration of voriconazole (VRZ). This study aimed to investigate the correlation between EMIT and high-performance liquid chromatography/ultraviolet rays (HPLC/UV) in determining the plasma VRZ trough concentration in children, in China. A total of 419 blood samples were collected, and plasma VRZ concentrations were detected by the EMIT and HPLC methods. The results of 304 samples were analysed after excluding samples that were undetectable or beyond the quantification limit. A test result value of 0 was defined as undetectable, while concentrations outside the detection range (0.2 − 20.0 μg/ml for HPLC and 0.5 − 16.0 µg/ml for EMIT) were defined as beyond the quantification limit. Results from both methods were compared using the Passing Bablok regression, Bland-Altman plot analysis, and paired Wilcoxon test. The plasma VRZ concentrations determined by EMIT and HPLC showed a strong linear correlation through the linear regression equation Y EMIT = 1.310 × HPLC +0.149 (R 2 = 0.9082). The Bland-Altman plot analysis showed poor level consistency as measured by the two methods. The paired Wilcoxon-test showed a significant difference between the two methods (p < .0001). Compared to EMIT, HPLC accurately detected plasma VRZ concentration, making it suitable for VRZ therapeutic drug monitoring. The numerical values of the EMIT-measured levels were higher than those of HPLC, which may be related to VRZ metabolites interference and co-administrated drugs.

Rapid and Simple Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) Method for Simultaneous Quantifications of Triazole Antifungals in Human Serum.

To develop and validate a one-step, rapid and simple reversed-phase high-performance liquid chromatography (HPLC)-based protocol for the simultaneous measurement of voriconazole (VCZ), posaconazole (POSA), itraconazole (ITC) in serum/plasma. Calibration standards (CS) and quality control samples were prepared in drug-free serum by spiking with the triazoles at different concentrations. HPLC was performed with C18 column, isocratic mobile phase after extraction with cold acetonitrile. The standardized method was tested in 2693 patients' serum/plasma samples. Linearity of CS for ITC, VCZ and POSA was proportional to the nominal concentration (correlation coefficient > 0.999). Limit of detection (mg/L) for ITC, VCZ and POSA was 0.25, 0.25 and 0.125, respectively. The lower limit of quantification (mg/L) for ITC, VCZ and POSA was 0.5, 0.5 and 0.25, respectively. Precision and accuracy were in acceptable range with 100% average percentage recovery. No interferences from endogenous substances and other antimicrobial compounds were noted. In clinical samples, the therapeutic range achieved for VCZ was 39.9%. Whereas, 61.1% and 44% of samples with ITC and POSA, respectively, were in the sub-therapeutic range. We developed a rapid and simple HPLC method to quantify common triazoles in a single chromatographic run allowing simultaneous measurement of different antifungals in a small volume of serum/plasma. Thus, therapeutic drug monitoring requests can be processed in one run without changing the protocol parameters, column or column conditioning thereby improving turnaround time.

Association of Voriconazole Trough Plasma Concentration with Efficacy and Incidence of Hepatotoxicity in Iranian Patients with Hematological Malignancies.

There are conflicting data regarding the association between plasma concentration of voriconazole (VCZ) and both efficacy and safety. This study investigates the association of VCZ trough plasma level with clinical efficacy and hepatotoxicity in the Iranian population suffering hematological malignancies. This cross-sectional study was performed on adult Iranian patients (age ≥ 18 years) with hematological malignancies undergoing treatment with oral or intravenous VCZ for proven or probable invasive aspergillosis. Plasma concentrations of VCZ were measured at two time points on day 4 and 14 during the study period. A total of 60 VCZ trough concentrations of 30 patients were drawn on days 4 and 14 after the initiation of treatment. There was no definite correlation between the mean plasma concentration of VCZ and VCZ dosage (p = 0.134, r = 0.280). In multivariable model, only plasma concentration of VCZ on day 14 was associated with the incidence of hepatotoxicity (p = 0.013; OR = 1.42, 95% CI = 1.07-3.24). Plasma trough concentration neither on day 4 nor on day 14 was related to the treatment response. No significant association was observed between the mean plasma concentration of VCZ and 3-month patients’ survival (p = 0.696). To conclude, VCZ trough concentration may not be a predictor of treatment response or 3-month patients’ survival. However, the wide inter- and intra-patient variability of VCZ plasma concentration coupled with the observed association between VCZ trough level and the incidence of hepatotoxicity would pose the question regarding the potential benefit of VCZ concentration monitoring.

Ubiquitous Distribution of Azole-Resistant Aspergillus fumigatus- Related Species in Outdoor Environments in Japan.

Aspergillus fumigatus-related species are responsible for causing aspergillosis, which is a fatal infectious disease. Recently, there has been a series of reports of A. fumigatus-related species that are resistant to azole drugs used in clinical practice for the treatment of fungal infections. Some of these species have been isolated from outdoor environments. Testing the drug susceptibility of the strains from outdoor environments, therefore, is important. In this study, we isolated and cultured 72 strains of A. fumigatus-related species from the outdoor environment in Japan. The isolates identified via morphological observation and molecular phylogenetic analysis were Aspergillus felis, Aspergillus lentulus, Aspergillus pseudoviridinutans, Aspergillus udagawae, and Aspergillus wyomingensis. The results of the drug susceptibility testing revealed that A. felis (6 of 14 strains) and A. pseudoviridinutans (13 of 17 strains) were resistant to itraconazole (ITCZ), with 4 mg/L or higher minimum inhibitory concentrations (MICs). The voriconazole (VRCZ)-resistant strains with 4 mg/L or higher MICs were A. felis (14 of 14), A. lentulus (4 of 4), A. pseudoviridinutans (15 of 17), A. udagawae (23 of 34), A. wyomingensis (1 of 3), and A. pseudoviridinutans (1 of 3). Among them, A. felis (1 of 14) and A. pseudoviridinutans (7 of 17) demonstrated 8 mg/L or higher MICs for ITCZ and VRCZ. These results indicate that A. fumigatus-related species resistant to ITCZ and VRCZ are widely distributed in outdoor environments in Japan.

Tailoring thixotropic mixed-lipid nanoconstructs of voriconazole for the management of Vulvovaginal candidiasis: Formulation, statistical optimization, in vitro characterization and in vivo assessment

Vulvovaginal candidiasis is a pervasive gynecological condition among women worldwide due to infection recurrence and resistance to conventional drugs. This calls for a novel formulation of alternative medication and with enhanced efficacy. This study aimed to fabricate mixed-lipid nanoconstructs (MLNCs) of voriconazole (VCZ) with a low concentration of lipids applying high shear homogenization and ultrasonication to form a semisolid formulation. Tefose 63 and Gelot 64 were employed as emulsifiers that are specified for vaginal preparations; as per their mucoadhesive properties and their texture enhancing characters, although usually used as lipids in different lipid carriers. A 24 factorial design was established and the optimized formulation was prepared using 10% total lipids, in which solid lipids (Sterotex NF: Glyceryl monostearate) ratio was 1.92:1 and the oils percentage was 30% (Maisine: Glyceryl monooleate, in the ratio of 1:1), and the emulsifiers mixture (Tefose 63: Gelot 64) ratio was 1:1, as 10% of total formulation weight. The optimized formulation with a viscosity of 964.49 ± 57.99 cp showed spherical nanoparticles (322.72 ± 15.11 nm) that entrapped 67.16 ± 3.45% of VCZ and exhibited release of 70.08 ± 2.87% in 8 h. The optimized formulation with high bioadhesive potentials significantly reduced the fungal burden in female Wistar rats infected with vaginal candidiasis, compared to the aqueous VCZ suspension (p < .05). Furthermore, in vivo histopathological findings proved the effectiveness and the safety of the optimized MLNCs formulation after vaginal application. Inclusively, MLNCs formulation could be a promising vaginal delivery system of VCZ for the treatment of vulvovaginal candidiasis.

In vitro antifungal susceptibility of yeasts and molds isolated from sputum of tuberculosis relapse and retreatment patients.

Introductionopportunistic fungal infections due to immunosuppression coupled with antifungal drug resistance are an emerging challenge globally. The present study examined the antifungal susceptibility of yeasts and molds from sputum of tuberculosis retreatment and relapse patients at selected reference facilities in Kenya.Methodsa total of 340 sputa samples from patients who gave written informed consent were examined. Fungal culture was done on sabouraud dextrose agar (SDA). Molds were identified by macroscopic and microscopic features while yeasts were inoculated on CHROMTMagar Candida and confirmed using API 20C AUXTM. Itraconazole (ICZ), voriconazole (VCZ), fluconazole (FCZ) and amphotericin B (AMB) were tested using broth micro-dilution methods according to Clinical and Laboratory Standards Institute (CLSI).Resultsout of the 340 samples, 14.4% (n=49) and 15.6% (n=53) were positive for yeasts and molds respectively. Candida albicans and C. krusei were the most predominant isolates constituting 49.0% (n=24) and 20.4% (n=10) of the total yeasts respectively. Aspergillus spp. were the most frequent (22.6%) molds and isolates with MICs ≥4μg/ml on the antifungal agents were noted. All the molds except two (n=2) isolates of Scedosporium aspiopermum exhibited MICs >4μg/ml for fluconazole. Overall, molds were more sensitive to AMB and VCZ. Candida albicans had MIC50 <0.06μg/ml, and MIC90<4μg/ml. There was a statistically significant difference (F=3.7, P=0.004<0.05) in the overall sensitivity pattern of molds for the four antifungal agents while there was no significant difference (F=1.7, P=0.154>0.05) in sensitivity exhibited by the yeasts.Conclusionthe study demonstrates the significance of fungal colonization in presumptive tuberculosis retreatment or relapse with evidence of triazole resistance. There is need to strengthen fungal diagnostic and clinical management capabilities in susceptible populations.

1149. Baseline Chest CT Findings in Patients with Pulmonary Mold Infections: A Post-Hoc Analysis from the Phase 3 SECURE Study that Compared Isavuconazole to Voriconazole for the Primary Treatment of Invasive Mold Disease

BackgroundSECURE was a global, double-blind, Phase 3 study that randomized 527 patients 1:1 to isavuconazole (ISAV) or voriconazole (VORI) for the primary treatment of invasive mold disease (IMD) caused by Aspergillus or other filamentous fungi. Patients were classified as having proven/probable IMD (PP-IMD) or possible IMD (PS-IMD) according to EORTC/MSG 2008 criteria, and the majority (n=412) had pulmonary disease only. This post-hoc analysis describes baseline CT findings in these patients and explores the association between these findings and treatment outcomes.MethodsA blinded, independent review committee assessed the certainty of diagnosis (PP-IMD vs PS-IMD), location of disease (pulmonary only, pulmonary plus other organ, non-pulmonary only), and both overall and clinical responses at end-of-treatment. Radiology assessments were done by central blinded radiologists who characterized pulmonary lesions as follows: well-defined nodule(s) with or without halo sign, wedge-shaped infiltrate, cavity, air crescent sign, or non-specific focal infiltrate.ResultsOf the 412 patients with pulmonary disease only, 223 (54%) had PP-IMD and 189 (46%) had PS-IMD. Well-defined nodule(s) was the predominant radiological finding at baseline in patients with PP-IMD or PS-IMD (PP-IMD 55%, PS-IMD 63%), followed by non-specific focal infiltrate (PP-IMD 43%, PS-IMD 41%), wedge-shaped infiltrate (PP-IMD 24%, PS-IMD 30%), and halo (PP-IMD 25%, PS-IMD 28%). A small proportion of patients had a cavity (PP-IMD 12%, PS-IMD 9%) or air crescent sign (PP-IMD 2%, PS-IMD 4%).Patients with air crescent sign had low all-cause mortality through Days 42 and 84, and high overall and clinical response rates at end-of treatment (PP-IMD: 0, 0, 75%, 100% and PS-IMD: 0, 13%, 75%, 88%, respectively). There was no other clear association between baseline CT findings and either outcomes of all-cause mortality or overall and clinical responses.Figure. Chest CT findings at baseline in patients with PP-IMD or PS-IMD (pulmonary only) ConclusionIn patients with pulmonary IMD, the predominant radiological finding at baseline was well-defined nodule(s) in both PP-IMD and PS-IMD. Air crescent sign was infrequent, but was associated with lower all-cause mortality and higher overall and clinical responses. Otherwise, baseline CT findings did not appear to predict treatment outcomes in this Phase 3 study.Disclosures Kamal Hamed, n/a, Basilea Pharmaceutica International Ltd. (Employee) Marc Engelhardt, n/a, Basilea Pharmaceutica International Ltd. (Board Member, Consultant, Employee, Grant/Research Support, Scientific Research Study Investigator, Advisor or Review Panel member, Research Grant or Support, Shareholder, Speaker’s Bureau, Independent Contractor, Other Financial or Material Support)Basilea Pharmaceutica International Ltd. (Employee) Mikael Saulay, n/a, Basilea Pharmaceutica International Ltd. (Employee) Laura Kovanda, n/a, Astellas Pharma Inc (Employee)

Comment on “is Phaeohyphomycosis an emerging fungal Infection in Iran? A Short Communication

Dear editor We recently read an interesting article entitled “Is Phaeohyphomycosis an emerging fungal Infection in Iran? A Short Communication” [1] which is published in your journal. This article discusses the prevalence of Phaeohyphomycosis cases in Iran. We published an article entitled “Cerebral Phaeohyphomycosis due to Rhinocladiella mackenziei in Persian Gulf region: A case and review” in 2018 [2]. Our case was a diabetic patient with the history of behcet's disease and with the diagnosis of brain abscesses due to Rhinocladiella mackenziei. For the patient we prescribed voriconazole at first then change the regimen to amphotericin b liposomal (5 mg/kg/day) with combination of posaconazole (200 mg every 6 hours). In the article published by zarrinfar et al recently [1], the authors have referenced our article [2] and instead of the original dose for posaconazole (200 mg every 6 hours), which we also had stated the dose in our article (200 mg every 6 hours), the authors have written wrong dose (200 mg daily) in the table. The dose of posaconazole for treatment is 200 mg every 6 hours and for prophylaxis is 200 mg every 8 hours. We request that this wrong dose be corrected in the table of article published in your journal.

Statistical optimization of hyaluronic acid enriched ultradeformable elastosomes for ocular delivery of voriconazole via Box-Behnken design: in vitro characterization and in vivo evaluation

Voriconazole (VCZ) is a well-known broad spectrum triazole antifungal, mainly used orally and intravenously. The study aimed to formulate VCZ into ultradeformable elastosomes for the topical treatment of ocular fungal keratitis. Different formulae were prepared using a modified ethanol injection method, employing a 33 Box-Behnken design. They were characterized by measuring their entrapment efficiency (EE%), particle size (PS), polydispersity index (PDI) and zeta potential (ZP). The optimized formula was subjected to further in vitro investigations and in vivo evaluation studies. The prepared vesicles had satisfactory EE%, PS, PDI and ZP values. The numerical optimization process suggested an optimal elastosomal formula (OE) composed of phosphatidyl choline and brij S100 at the weight ratio of 3.62: 1, 0.25%w/v hyaluronic acid and 5% (percentage from phosphatidyl choline/brij mixture) polyvinyl alcohol. It had high EE (72.6%), acceptable PS and PDI (362.4 nm and 0.25, respectively) and highly negative ZP of −41.7 mV. OE exhibited higher elasticity than conventional liposomes, with acceptable stability for three months. Transmission electron microscopy demonstrated the spherical morphology of vesicles with an external transparent coat of Hyaluronic acid. OE was expected to cause no ocular irritation or blurring in vision as reflected by pH and refractive index measurements. The histopathological study revealed the safety of OE for ocular use. The fungal susceptibility testing using Candida albicans demonstrated the superiority of OE to VCZ suspension, with greater and more durable growth inhibition. Therefore, OE can be regarded as a promising formula, achieving both safety and efficacy.

Voriconazole Ternary Micellar Systems for the Treatment of Ocular Mycosis: Statistical Optimization and InVivo Evaluation.

Voriconazole (VRC) is a broad spectrum, second generation triazole antifungal. The main use of VRC is via the oral and intravenous route. The study aimed to formulate VRC into ternary micellar systems (TMSs) for the topical treatment of ocular mycosis. TMSs were successfully prepared by water addition/solvent evaporation method, applying a 3-factor D-optimal design. The numerical optimization process suggested an optimal formula (OTMS) composed of total Pluronics to drug weight ratio of 22.89: 1, 1:1 weight ratio of Pluronic® P123 and F68, and 2% w/v of Labrasol. OTMS had high solubilization efficiency of 98.0%, small micellar size of 21.8 nm and suitable zeta potential and polydispersity index values of -9.0 mV and 0.261, respectively. OTMS exhibited acceptable stability for 3months. Transmission electron microscopy demonstrated the spherical morphology of micelles. OTMS was expected to cause no ocular irritation or blurring in vision as reflected by pH and refractive index measurements. The histopathological study revealed the safety of OTMS for ocular use. The fungal susceptibility testing using Candida albicans demonstrated the superiority of OTMS to VRC suspension, with greater and more durable growth inhibition. Therefore, ocular application of optimized VRC-loaded TMSs can be a promising treatment for ocular mycosis.

Tpo3 and dur3, Aspergillus fumigatus Plasma Membrane Regulators of Polyamines, Regulate Polyamine Homeostasis and Susceptibility to Itraconazole.

Aspergillus fumigatus is a well-known opportunistic pathogen that causes invasive aspergillosis (IA) infections, which have high mortality rates in immunosuppressed individuals. Long-term antifungal drug azole use in clinical treatment and agriculture results in loss of efficacy or drug resistance. Drug resistance is related to cellular metabolites and the corresponding gene transcription. In this study, through untargeted metabolomics and transcriptomics under itraconazole (ITC) treatment, we identified two plasma membrane-localized polyamine regulators tpo3 and dur3, which were important for polyamine homeostasis and susceptibility to ITC in A. fumigatus. In the absence of tpo3 and/or dur3, the levels of cytoplasmic polyamines had a moderate increase, which enhanced the tolerance of A. fumigatus to ITC. In comparison, overexpression of tpo3 or dur3 induced a drastic increase in polyamines, which increased the sensitivity of A. fumigatus to ITC. Further analysis revealed that polyamines concentration-dependently affected the susceptibility of A. fumigatus to ITC by scavenging reactive oxygen species (ROS) at a moderate concentration and promoting the production of ROS at a high concentration rather than regulating drug transport. Moreover, inhibition of polyamine biosynthesis reduced the intracellular polyamine content, resulted in accumulation of ROS and enhanced the antifungal activity of ITC. Interestingly, A. fumigatus produces much lower levels of ROS under voriconazole (VOC) treatment than under ITC-treatment. Accordingly, our study established the link among the polyamine regulators tpo3 and dur3, polyamine homeostasis, ROS content, and ITC susceptibility in A. fumigatus.

Effects of the Glass-Forming Ability and Annealing Conditions on Cocrystallization Behaviors via Rapid Solvent Removal: A Case Study of Voriconazole.

The kinetic entrapment of molecules in an amorphous phase is a common obstacle to cocrystal screening using rapid solvent removal, especially for drugs with a moderate or high glass-forming ability (GFA). The aim of this study was to elucidate the effects of the coformer’s GFA and annealing conditions on the nature of amorphous phase transformation to the cocrystal counterpart. Attempts were made to cocrystallize voriconazole (VRC) with four structural analogues, namely fumaric acid (FUM), tartaric acid (TAR), malic acid (MAL), and maleic acid (MAE). The overall GFA of VRC binary systems increased with decreasing glass transition temperatures (Tgs) of these diacids, which appeared as a critical parameter for predicting the cocrystallization propensity such that a high-Tg coformer is more desirable. A new 1:1 VRC-TAR cocrystal was successfully produced via a supercooled-mediated re-cocrystallization process, and characterized by PXRD, DSC, and FTIR. The cocrystal purity against the annealing temperature displayed a bell-shaped curve, with a threshold at 40 °C. The isothermal phase purity improved with annealing and adhered to the Kolmogorov–Johnson–Mehl–Avrami kinetics. The superior dissolution behavior of the VRC-TAR cocrystal could minimize VRC precipitation upon gastric emptying. This study offers a simple but useful guide for efficient cocrystal screening based on the Tg of structurally similar coformers, annealing temperature, and time.

Optimization of voriconazole therapy for treatment of invasive aspergillosis: Pharmacogenomics and inflammatory status need to be evaluated.

Cytochrome 2C19 genotype-directed dosing of voriconazole (VRC) reduces the incidence of insufficient VRC trough concentrations (Cmin ) but does not account for CYP3A polymorphisms, also involved in VRC metabolism. This prospective observational study aimed to evaluate the utility of a genetic score combining CYP2C19 and CYP3A genotypes to predict insufficient initial VRC Cmin (<1mg/L). The genetic score was determined in hematological patients treated with VRC. The higher the genetic score, the faster the metabolism of the patient. The impact of the genetic score was evaluated considering initial VRC Cmin and all VRC Cmin (n=159) determined during longitudinal therapeutic drug monitoring. Forty-three patients were included, of whom 41 received VRC for curative indication. Thirty-six patients had a genetic score ≥2, of whom 11 had an initial insufficient VRC Cmin . A genetic score ≥2 had a positive predictive value of 0.31 for having an initial insufficient VRC Cmin and initial VRC Cmin was not associated with the genetic score. The lack of association between the genetic score and VRC Cmin may be related to the inflammatory status of the patients (C-reactive protein [CRP] levels: median [Q1-Q3]: 43.0 [11.0-110.0]mg/L), as multivariate analysis performed on all VRC Cmin identified CRP as an independent determinant of the VRC Cmin adjusted for dose (P<.0001). The combined genetic score did not predict low VRC exposure in patients with inflammation, which is frequent in patients with invasive fungal infections. Strategies for the individualization of VRC dose should integrate the inflammatory status of patients in addition to pharmacogenetic variants.

Transition of triazole-resistant Aspergillus fumigatus isolates in a Japanese tertiary hospital and subsequent genetic analysis

ObjectiveTo evaluate the annual variation in the frequency of patient-acquired azole-resistant Aspergillus fumigatus (ARAf), and correlate it to the amount of oral triazole prescribed, in Nagasaki, Japan. MethodsA. fumigatus isolates from respiratory specimens collected in the Nagasaki University Hospital (NUH) between 1996 and 2017 were included in the study. The amount of oral triazole prescribed in NUH since 2001 was obtained from the medical ordering system. Mutations in cyp51A, hmg1, and erg6 genes of ARAf were also analysed. ResultsFrom a total of 240 ARAf strains, 12 (5%), 6 (2.5%), 15 (6.25%), and 3 (1.25%) strains were resistant to itraconazole (ITC), voriconazole (VRC), to either ITC or VRC, and both triazoles, respectively. The amount of prescribed VRC increased annually, and was three times as large as that of ITC in 2017. All eleven patients harbouring ITC-resistant strains had a history of prior ITC treatment, while only one of six patients harbouring VRC-resistant strains had a history of prior VRC treatment. cyp51A mutations were recorded in 10 strains; however, tandem repeat mutations of the promoter region of cyp51A were not observed. Several azole-resistant strains had non-cyp51A mutations. ConclusionsThe frequency of patient-acquired ARAf is not increasing in Nagasaki, Japan. Furthermore, the prevalence of VRC-induced ARAf was rare despite the remarkable increase in the amount of prescribed VRC. Mutations in genes other than cyp51A should also be considered when ARAf strains are obtained from patients treated with azole antifungals.