Bioisosteric modification on benzylidene-carbonyl compounds improved the drug-likeness and maintained the antifungal activity against Sporothrix brasiliensis.

Considering the emergence of antifungal resistance on Sporothrix brasiliensis, we aimed to assess new benzylidene-carbonyl compounds against feline-borne S. brasiliensis isolates. The compounds were designed as bioisosteres from previously reported benzylidene-ketones generating the p-coumaric (1), cinnamic (2), p-methoxycinnamic (3) and caffeic acid (4) analogues. The corresponding compounds were tested against feline isolates of S. brasiliensis with sensitivity (n = 4) and resistance (n = 5) to itraconazole (ITZ), following the M38-A2 protocol (CLSI, Reference method for broth dilution antifungal susceptibility testing of filamentous fungi M38-A2 Guideline, 2008). Eleven analogues showed activity against all fungal strains with minimum inhibitory concentrations (MIC) ≤1 mg/ml (1a-d, 2e, 3b, 3e, 4, 4a and 5e) and fungicidal concentrations (MFC) ≤1 mg/ml (1b, 1d, 3e and 4a), whereas 3 was the less active with both MIC and MFC values above 1 mg/ml. Compound 3e (4-methoxy-N-butylcinnamamide) was the most potent (MICrange 0.08-0.16 mg/ml; MFCrange 0.32-0.64 mg/ml) from the set, suggesting a different role of the substituents in ester and amide derivatives. The designed compounds proved to be important prototypes with improved drug-likeness to achieve compounds with higher activity against ITZ-resistant S. brasiliensis.

Synthetic peptides against Trichophyton mentagrophytes and T. rubrum: Mechanisms of action and efficiency compared to griseofulvin and itraconazole.

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.

Formulation and evaluation of itraconazole liposomes for Hedgehog pathway inhibition.

Itraconazole (ITZ) is an FDA-approved antifungal agent that has recently been explored for novel biological properties. In particular, ITZ was identified as a potent inhibitor of the hedgehog (Hh) pathway, a cell signalling pathway that has been linked to a variety of cancers and accounts for ∼25% of paediatric medulloblastoma (MB) cases. To date, there is not a targeted therapeutic option for paediatric MB, resulting in long-term side effects such as hormone deficiency, organ damage and secondary cancers. A primary obstacle for developing targeted therapy for brain ailments is the presence of the blood-brain barrier (BBB), which protects the brain from potentially harmful substances. Due to its size and hydrophobicity, ITZ does not penetrate the BBB. Alternatively, liposomes are being increasingly used within the clinic to increase drug bioavailability, target specificity and BBB permeability. With this in mind, we have successfully developed ITZ-containing liposomes with an optimal size for BBB penetration (<100 nm) and encapsulation efficiency (∼95%) by utilizing a continuous manufacturing approach-turbulent coaxial jet in co-flow. Our preliminary in vitro data demonstrate that these liposomes inhibit the Hh pathway, albeit at a reduced level in comparison to free ITZ. (196/250 words).

Utility of Films to Anticipate Effect of Drug Load and Polymer on Dissolution Performance from Tablets of Amorphous Itraconazole Spray-Dried Dispersions.

Because spray-dried dispersion (SDD) performance depends on polymer selection and drug load, time- and resource-sparing methods to screen drug/polymer combinations before spray drying are desirable. The primary objective was to assess the utility of films to anticipate the effects of drug load and polymer grade on dissolution performance of tablets containing SDDs of itraconazole (ITZ). A secondary objective was to characterize the solid-state attributes of films and SDDs to explain drug load and polymer effects on dissolution performance. SDDs employed three different grades of hypromellose acetate succinate (i.e., either HPMCAS-L, HPMCAS-M, or HPMCAS-H). Solid-state characterization employed differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), and solid-state nuclear magnetic resonance (ssNMR) spectroscopy. Results indicate that films correctly anticipated the effects of drug load and polymer on dissolution performance. The best dissolution profiles were observed under the following conditions: 20% drug loading performed better than 30% for both films and SDDs, and the polymer grade rank order was HPMCAS-L > HPMCAS-M > HPMCAS-H for both films and SDDs. No dissolution was detected from films or SDDs containing HPMCAS-H. Solid-state characterization revealed percent crystallinity and phase miscibility as contributing factors to dissolution, but were not the sole factors. Amorphous content in films varied with drug load (10% > 20% > 30%) and polymer grades (HPMCAS-L > HPMCAS-M > HPMCAS-H), in agreement with dissolution. In conclusion, films anticipated the rank-order effects of drug load and polymer grade on dissolution performance from SDDs of ITZ, in part through percent crystallinity and phase miscibility influences.

Proof-of-concept preparation and characterization of dual-drug amorphous nanoparticle complex as fixed-dose combination of poorly soluble drugs.

OBJECTIVES: To carry out a proof-of-concept study on the development of dual-drug amorphous nanoparticle complex (nanoplex in short) as a potential formulation platform for fixed-dose combination (FDC) of poorly-soluble drugs. SIGNIFICANCE: FDC has been proven effective in improving patient compliance for treatment that requires complex multidrug regimen. Currently, there is growing interest to develop FDC of poorly-soluble drugs due to the increased number of drugs exhibiting poor solubility thus low bioavailability. METHODS: The dual-drug nanoplex was prepared by electrostatically-driven co-complexation of drug molecules with oppositely charged dextran sulfate, using ciprofloxacin (CIP) and itraconazole (ITZ) as the model poorly-soluble drugs. RESULTS: We first verified that the co-complexation products were dual-drug CIP-ITZ nanoplex, and not binary mixtures of the single-drug CIP and ITZ nanoplexes, by demonstrating their distinct thermal behaviors and dissolution characteristics. Depending on the preparation condition, the dual-drug nanoplex exhibited size and zeta potential of 160-410 nm and -35-50 mV, respectively. The individual drug payloads were readily manipulated by varying the CIP/ITZ mass ratio in the feed, resulting in CIP and ITZ payloads in the range of 60-30% and 15-45%, respectively. The CIP-ITZ nanoplex, however, exhibited diminished CIP supersaturation generation, thus lower CIP solubility enhancement, compared to the single-drug CIP nanoplex. The CIP-ITZ nanoplex, nonetheless, remained capable of generating high ITZ supersaturation level. CONCLUSION: Dual-drug nanoplex was successfully prepared with a high degree of control over its physical characteristics. Nevertheless, whether dual-drug nanoplex always exhibits diminished solubility enhancement compared to its single-drug counterparts needs to be investigated using different poorly-soluble drugs.

Fluorescent Tracking of the Endoplasmic Reticulum in Live Pathogenic Fungal Cells.

In fungal cells, the endoplasmic reticulum (ER) harbors several of the enzymes involved in the biosynthesis of ergosterol, an essential membrane component, making this organelle the site of action of antifungal azole drugs, used as a first-line treatment for fungal infections. This highlights the need for specific fluorescent labeling of this organelle in cells of pathogenic fungi. Here we report on the development and evaluation of a collection of fluorescent ER trackers in a panel of Candida, considered the most frequently encountered pathogen in fungal infections. These trackers enabled imaging of the ER in live fungal cells. Organelle specificity was associated with the expression of the target enzyme of antifungal azoles that resides in the ER; specific ER labeling was not observed in mutant cells lacking this enzyme. Labeling of live Candida cells with a combination of a mitotracker and one of the novel fungal ER trackers revealed sites of contact between the ER and mitochondria. These fungal ER trackers therefore offer unique molecular tools for the study of the ER and its interactions with other organelles in live cells of pathogenic fungi.

Development of Fast-Dissolving Amorphous Solid Dispersion of Itraconazole by Melt Extrusion of its Mixture with Weak Organic Carboxylic Acid and Polymer.

PURPOSE: The purpose of this study was to explore the feasibility of developing amorphous solid dispersion (ASD) by inducing acid-base interaction at an elevated temperature using hot melt extrusion. METHODS: Itraconazole and glutaric acid, which do not form salt with each other, were selected as, respectively, model basic drug and weak organic acid. A 1:4:1w/w mixture of itraconazole, glutaric acid and a polymer, Kollidon®VA64, was melt extruded at 95°C. The ground extrudate was characterized by DSC and PXRD and then tested for dissolution at pH 1.2, followed by a change in pH to 5.5. RESULTS: Despite the high melting point of 168°C, itraconazole dissolved in glutaric acid at around the melting temperature of acid (~98°C), and physically stable ASD was produced when the formulation was extruded at 95°C. Capsules containing 100-mg equivalent of itraconazole dissolved rapidly at pH 1.2 producing highly supersaturated solution. When the pH was changed from 1.2 to 5.5, very fine suspensions, facilitated by the presence of Kollidon®VA64, was formed. CONCLUSIONS: Physically stable ASD of itraconazole with high drug load was prepared by interaction with glutaric acid in a hot melt extruder. This may be used as a platform technology for the development ASD of most poorly water-soluble basic drugs.

Preparation and evaluation of dual-mode floating gastroretentive tablets containing itraconazole.

The aims of the present study were to prepare new dual-mode floating gastroretentive tablets (DF-GRT) containing itraconazole (ITR) and to evaluate influence of the dosage forms on pharmacokinetic parameters of ITR. The solubility of ITR was enhanced around 200 times (from 1.54 to 248.38 µg/mL) by preparing solid dispersion (SD) with hydroxypropylmethyl cellulose. Buoyancy of DF-GRT containing ITR-SD was established by both camphor sublimation and gas generation. Camphor sublimation decreased density of DF-GRT by making pores in tablet matrix, which led to elimination of lag time for floating. Carbon dioxide generated by sodium bicarbonate and citric acid helped to maintain buoyancy of DF-GRT. Therefore DF-GRT floated on the medium without lag time until disintegrated entirely during in vitro release study. They released 89.11% of the drug at 2 h. Residual camphor was <0.5 wt% after sublimation. The pharmacokinetics of DF-GRT was evaluated in six miniature pigs and compared to immediate release tablets (IRT). Mean AUC ratio of GRT/IRT was 1.36 but there was no statistical difference between AUC values. However delayed tmax, increased MRT and equivalent Cmax of DF-GRT supposed it could be a promising tool for gastroretentive drug delivery system containing ITR.

Influence of formulation and processing variables on properties of itraconazole nanoparticles made by advanced evaporative precipitation into aqueous solution.

Nanoparticles, of the poorly water-soluble drug, itraconazole (ITZ), were produced by the Advanced Evaporative Precipitation into Aqueous Solution process (Advanced EPAS). This process combines emulsion templating and EPAS processing to provide improved control over the size distribution of precipitated particles. Specifically, oil-in-water emulsions containing the drug and suitable stabilizers are sprayed into a heated aqueous solution to induce precipitation of the drug in form of nanoparticles. The influence of processing parameters (temperature and volume of the heated aqueous solution; type of nozzle) and formulation aspects (stabilizer concentrations; total solid concentrations) on the size of suspended ITZ particles, as determined by laser diffraction, was investigated. Furthermore, freeze-dried ITZ nanoparticles were evaluated regarding their morphology, crystallinity, redispersibility, and dissolution behavior. Results indicate that a robust precipitation process was developed such that size distribution of dispersed nanoparticles was shown to be largely independent across the different processing and formulation parameters. Freeze-drying of colloidal dispersions resulted in micron-sized agglomerates composed of spherical, sub-300-nm particles characterized by reduced crystallinity and high ITZ potencies of up to 94% (w/w). The use of sucrose prevented particle agglomeration and resulted in powders that were readily reconstituted and reached high and sustained supersaturation levels upon dissolution in aqueous media.

Ditosylate salt of itraconazole and dissolution enhancement using cyclodextrins.

Salt formation has been a promising approach for improving the solubility of poorly soluble acidic and basic drugs. The aim of the present study was to prepare the salt form of itraconazole (ITZ), a hydrophobic drug to improve the solubility and hence dissolution performance. Itraconazolium ditolenesulfonate salt (ITZDITOS) was synthesized from ITZ using acid addition reaction with p-toluenesulfonic acid. Salt characterization was performed using (1)H NMR, mass spectrometry, Fourier transform infrared spectroscopy, differential scanning calorimetry, and X-ray diffraction. The particle size and morphology was studied using dynamic light scattering technique and scanning electron microscopy, respectively. The solubility of the salt in water and various pharmaceutical solvents was found multifold than ITZ. The dissolution study exhibited 5.5-fold greater percentage release value in 3 h of ITZDITOS (44.53%) as compared with ITZ (8.54%). Results of in vitro antifungal studies using broth microdilution technique indicate that ITZDITOS possessed similar antifungal profile as that of ITZ when tested against four fungal pathogens. Furthermore, the physical mixtures of ITZDITOS with two cyclodextrins, ß-cyclodextrin (ß-CD), and 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) were prepared in different molar ratios and were evaluated for in vitro release. It was observed that in only 30 min of dissolution study, about 74 and 81% of drug was released from 1:3 molar ratios of ITZDITOS with ß-CD and ITZDITOS with HP-ß-CD, respectively, which was distinctly higher than the drug released from ITZ commercial capsules (70%). The findings warrant further preclinical and clinical studies on ITZDITOS so that it can be established as an alternative to ITZ for developing oral formulations.

Itraconazole side chain analogues: structure-activity relationship studies for inhibition of endothelial cell proliferation, vascular endothelial growth factor receptor 2 (VEGFR2) glycosylation, and hedgehog signaling.

Itraconazole is an antifungal drug that was recently found to possess potent antiangiogenic activity and anti-hedgehog (Hh) pathway activity. To search for analogues of itraconazole with greater potency and to understand the structure-activity relationship in both antiangiogenic and Hh targeting activity, 25 itraconazole side chain analogues were synthesized and assayed for inhibition of endothelial cell proliferation and Gli1 transcription in a medulloblastoma (MB) culture. Through this analysis, we have identified analogues with increased potency for inhibiting endothelial cell proliferation and the Hh pathway, as well as VEGFR2 glycosylation that was recently found to be inhibited by itraconazole. An SAR analysis of these activities revealed that potent activity of the analogues against VEGFR2 glycosylation was generally driven by side chains of at least four carbons in composition with branching at the α or ß position. SAR trends for targeting the Hh pathway were divergent from those related to HUVEC proliferation or VEGFR2 glycosylation. These results also suggest that modification of the sec-butyl side chain can lead to enhancement of the biological activity of itraconazole.

Design and synthesis of pyridine-substituted itraconazole analogues with improved antifungal activities, water solubility and bioavailability.

To improve antifungal activities, water solubility and bioavailability, a series of novel analogues of itraconazole-containing pyridine rings were designed and synthesized. Their antifungal activities were evaluated in vitro against six clinically important fungi by measuring the minimal inhibitory concentrations (MICs). Most of the compounds showed more potent antifungal activities than that of itraconazole. In particular, the analogues 30d, 30c, 31c, and 36d exhibited much higher solubility and bioavailability than that of itraconazole. The bioavailability of 36d (42.2%) was five times higher than that of itraconazole (8%) and was negative for genetic toxicology in the Ames test.

Comparative study of itraconazole-cyclodextrin inclusion complex and its commercial product.

Itraconazole (ITZ) solid complex using hydroxypropyl-beta-cyclodextrin (ITZ-HP-beta-CD) with 20% polyvinylpyrrolidone was prepared by a co-evaporation method. The complex improved antifungal activity against C. parapasilosis and C. albicans. The complex demonstrated good flow and compressibility characteristics. The complex was formulated as a capsule dosage form and drug release was evaluated. Capsules containing ITZ-HP-beta-CD at a molar ratio of 1:3 with 20% polyvinylpyrrolidone have a faster dissolution rate than commercial capsules (Sporanox). About 88% of ITZ was released in less than 30 min and the initial dissolution rate exhibited a 3.5-fold increase compared to the commercial product. UV spectrophotometeric, HPLC, and antimicrobial methods were used to determine ITZ concentration in the release medium and the results obtained by these methods are reported. It was found that HPLC analysis is a suitable and reliable method for determination of the drug concentration with a coefficient of variation less than 10%. The intraday precision showed a coefficient of variation less than 3.96%, and that for interday was less than 4.99%. The HPLC method was more accurate and precise than the antimicrobial and UV-spectrophotometric methods for determination of ITZ concentration present in the release medium.

Carbon analogs of antifungal dioxane-triazole derivatives: synthesis and in vitro activities.

A new series of triazole compounds possessing a carbon atom in place of a sulfur atom were efficiently synthesized and their in vitro antifungal activities were investigated. The carbon analogs showed excellent in vitro activity against Candida, Cryptococcus, and Aspergillus species. The MICs of compound 1c against C. albicans ATCC24433, C. neoformans TIMM1855, and A. fumigatus ATCC26430 were 0.016, 0.016, and 0.125 microg/mL, respectively (MICs of fluconazole: 0.5, >4, and >4 microg/mL; MICs of itraconazole: 0.125, 0.25, and 0.25 microg/mL).

Clinical study of solid dispersions of itraconazole prepared by hot-stage extrusion.

The aim of this study was to investigate the performance of three new solid dispersion formulations of itraconazole in human volunteers in comparison with Sporanox, the marketed form. Solid dispersions made up of itraconazole (40%, w/w) and HPMC 2910, Eudragit E100 or a mixture of Eudragit E100-PVPVA64 were manufactured by hot-stage extrusion and filled in gelatin capsules. The formulations were tested in eight human volunteers in a double blind, single dose, and cross-over study. Concentrations of the drug and its metabolite hydroxyitraconazole in the plasma were determined using HPLC. The in vivo performance was evaluated by comparing the mean area under the plasma concentration-time curves (AUC), the mean maximum plasma concentration (C(max)), and the mean time to reach C(max) (T(max)). The mean bioavailability of itraconazole was comparable after administration of the HPMC solid dispersion, compared to Sporanox, while it was lower after administration of the Eudragit E100 or Eudragit E100-PVPVA64 dispersions. Due to high variability, a significant decrease in AUC and C(max) was only observed for the Eudragit E100-PVPVA formulation. Although the solid dispersions showed different in vitro dissolution behaviour, T(max) values were comparable. The same observations with respect to AUC, C(max) and T(max) could be made for hydroxyitraconazole. The present results indicate that hot-stage extrusion can be considered as a valuable alternative for manufacturing solid dispersions of itraconazole.

Itraconazole formation using supercritical carbon dioxide.

A new method of preparing Itraconazole (C35H38Cl2N8O4), a synthetic triazole antifungal agent, was developed using supercritical carbon dioxide (SC CO2) while eliminating the use of toxic solvents. Dissolution amounts of the product were measured in gastric fluid and compared to those of conventional drug formulations. Different operating conditions (five levels of treatment temperature ranging between 110-140 degrees C, four levels of treatment pressure ranging between 30-400 atm, and four different treatment times ranging from 10-60 minutes) were tested in order to produce a desired Itraconazole product, which does not degrade during the product formation and has the highest extent of dissolution in gastric fluid after one hour. Itraconazole dissolution of 100% at one-hour was achieved for the drug produced at the optimum treatment condition: 135 degrees C, 300 atm, and 30 minutes. Extent of dissolution obtained from this solvent and detergent-free process is 10% higher than that of the conventional method involving toxic organic solvents. Itraconazole produced using SC CO2 should provide minimal side effects in human body.

Preparation and characterization of nanofibers containing amorphous drug dispersions generated by electrostatic spinning.

PURPOSE: We assessed the application of water-soluble polymer-based nanofibers prepared by electrostatic spinning as a means of altering the dissolution rate of the poorly water-soluble drug, itraconazole. METHODS: Organic solvent-based solutions of itraconazole/HPMC mixtures were electrostatically spun at 16 and 24 kV. The formed nanofibers were collected as a non-woven fabric. The samples were analyzed by scanning electron microscopy. differential scanning calorimetry, and dissolution rate. RESULTS: Scanning electron microscopy showed fiber diameters of 1-4 microm and 300-500 nm depending on the applied voltage. Differential scanning calorimetry measurements found that the melting endotherm for itraconazole was not present, suggesting the formation of an amorphous solid dispersion or solution. Dissolution studies assessed several presentations including direct addition of the non-woven fabrics to the dissolution vessels, folding weighed samples of the materials into hard gelatin capsules and placing folded material into a sinker. Controls included a physical mixture as well as solvent cast and melt extruded samples. Electrospun samples dissolved completely over time with the rate of dissolution depending on the formulation presentation and drug to polymer ratio. The physical mixture did not appreciably dissolve in these conditions. CONCLUSIONS: The application of electrostatic spinning to pharmaceutical applications resulted in dosage forms with useful and controllable dissolution properties.