Effects of the Hedgehog Signaling Inhibitor Itraconazole on Developing Rat Ovaries.

Early ovary development is considered to be largely hormone independent; yet, there are associations between fetal exposure to endocrine disrupting chemicals and reproductive disorders in women. This can potentially be explained by perturbations to establishment of ovarian endocrine function rather than interference with an already established hormone system. In this study we explore if Hedgehog (HH) signaling, a central pathway for correct ovary development, can be disrupted by exposure to HH-disrupting chemicals, using the antifungal itraconazole as model compound. In the mouse Leydig cell line TM3, used as a proxy for ovarian theca cells, itraconazole exposure had a suppressing effect on genes downstream of HH signaling, such as Gli1. Exposing explanted rat ovaries (gestational day 22 or postnatal day 3) to 30 µM itraconazole for 72 h induced significant suppression of genes in the HH signaling pathway with altered Ihh, Gli1, Ptch1, and Smo expression similar to those previously observed in Ihh/Dhh knock-out mice. Exposing rat dams to 50 mg/kg bw/day in the perinatal period did not induce observable changes in the offspring's ovaries. Overall, our results suggest that HH signal disruptors may affect ovary development with potential long-term consequences for female reproductive health. However, potent HH inhibitors would likely cause severe teratogenic effects at doses lower than those causing ovarian dysgenesis, so the concern with respect to reproductive disorder is for the presence of HH disruptors at low concentration in combination with other ovary or endocrine disrupting compounds.

Species-specific differences in the inhibition of 11ß-hydroxysteroid dehydrogenase 2 by itraconazole and posaconazole.

11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) converts active 11ß-hydroxyglucocorticoids to their inactive 11-keto forms, thereby preventing inappropriate mineralocorticoid receptor activation by glucocorticoids. Disruption of 11ß-HSD2 activity by genetic defects or inhibitors causes the syndrome of apparent mineralocorticoid excess (AME), characterized by hypokalemia, hypernatremia and hypertension. Recently, the azole antifungals itraconazole and posaconazole were identified to potently inhibit human 11ß-HSD2, and several case studies described patients with acquired AME. To begin to understand why this adverse drug effect was missed during preclinical investigations, the inhibitory potential of itraconazole, its main metabolite hydroxyitraconazole (OHI) and posaconazole against 11ß-HSD2 from human and three commonly used experimental animals was assessed. Whilst human 11ß-HSD2 was potently inhibited by all three compounds (IC50 values in the nanomolar range), the rat enzyme was moderately inhibited (1.5- to 6-fold higher IC50 values compared to human), and mouse and zebrafish 11ß-HSD2 were very weakly inhibited (IC50 values above 7 µM). Sequence alignment and application of newly generated homology models for human and mouse 11ß-HSD2 revealed significant differences in the C-terminal region and the substrate binding pocket. Exchange of the C-terminus and substitution of residues Leu170,Ile172 in mouse 11ß-HSD2 by the corresponding residues His170,Glu172 of the human enzyme resulted in a gain of sensitivity to itraconazole and posaconazole, resembling human 11ß-HSD2. The results provide an explanation for the observed species-specific 11ß-HSD2 inhibition by the studied azole antifungals. The obtained structure-activity relationship information should facilitate future assessments of 11ß-HSD2 inhibitors and aid choosing adequate animal models for efficacy and safety studies.

Comparative omics analyses of hepatotoxicity induced by oral azole drugs in mice liver and primary hepatocytes.

Ketoconazole (KTZ) and itraconazole (ITZ) are antifungal agents that have a broad spectrum of activity against fungal pathogens. However, the therapeutic indications of many antifungal drugs, including those of the azole group, are restricted due to possible hepatotoxicity. We performed toxicogenomic analyses using in vivo and in vitro models to investigate the molecular mechanisms underlying the hepatotoxicity of two azole antifungal drugs. C57BL/6 male mice were treated daily with KTZ or ITZ, sacrificed at days 1 or 7, and the serum biochemistry and histopathology results showed that the KTZ-treated mice exhibited hepatotoxicity. Primary hepatocytes from C57BL/6 mice also exposed to KTZ or ITZ, and the cytotoxic effects of KTZ and ITZ were evaluated; KTZ exerted a greater cytotoxic effect than ITZ. The gene expression profiles in the livers of the 7-day-treated group and primary hepatocytes of the 24-h-treated group for both KTZ and ITZ were comparatively analyzed. Differentially expressed genes were selected based on the fold-changes and statistical significance, and the biological functions were analyzed using ingenuity pathways analysis. The results revealed that genes related to cholesterol synthesis were overexpressed in the liver in the KTZ-treated group, whereas expression of those related to acute phase injury was significantly altered in the ITZ-treated group. Causal gene analyses suggested that sterol regulatory element-binding transcription factors are key regulators that activate the transcription of target genes associated with the hepatotoxicity induced by oral KTZ. These findings enhance our understanding of the molecular mechanisms underlying the hepatotoxicity of azole drugs.

Divergence of Antiangiogenic Activity and Hepatotoxicity of Different Stereoisomers of Itraconazole.

PURPOSE: Itraconazole is a triazole antifungal drug that has recently been found to inhibit angiogenesis. Itraconazole is a relatively well-tolerated drug but shows hepatotoxicity in a small subset of patients. Itraconazole contains three chiral centers and the commercial itraconazole is composed of four cis-stereoisomers (named IT-A, IT-B, IT-C, and IT-D). We sought to determine whether the stereoisomers of itraconazole might differ in their antiangiogenic activity and hepatotoxicity. EXPERIMENTAL DESIGN: We assessed in vitro antiangiogenic activity of itraconazole and each stereoisomer using human umbilical vein endothelial cell (HUVEC) proliferation and tube formation assays. We also determined their hepatotoxicity using primary human hepatocytes in vitro and a mouse model in vivo Mouse Matrigel plug and tumor xenograft models were used to evaluate in vivo antiangiogenic and antitumor activities of the stereoisomers. RESULTS: Of the four stereoisomers contained in commercial itraconazole, we found that IT-A (2S,4R,2'R) and IT-C (2S,4R,2'S) were more potent for inhibition of angiogenesis than IT-B (2R,4S,2'R) and IT-D (2R,4S,2'S). Interestingly, IT-A and IT-B were more hepatotoxic than IT-C and IT-D. In mouse models, IT-C showed more potent antiangiogenic/antitumor activity with lower hepatotoxicity compared with itraconazole and IT-A. CONCLUSIONS: These results demonstrate the segregation of influence of stereochemistry at different positions of itraconazole on its antiangiogenic activity and hepatotoxicity, with the 2 and 4 positions affecting the former and the 2' position affecting the latter. They also suggest that IT-C may be superior to the racemic mixture of itraconazole as an anticancer drug candidate due to its lower hepatotoxicity and improved antiangiogenic activity. Clin Cancer Res; 22(11); 2709-20. ©2016 AACR.

Toxicidad de agentes antiparasitarios, antimicrobianos e insecticidas sobre larvas del camarón salino Artemia franciscana (Crustacea: Artemiidae) / Toxicity of antiparasitic, antimicrobial agents and insecticides on larvae of brine shrimp Artemia franciscana (crustacea: artemiidae)

Artemia franciscana "camarón salino", es un crustáceo sensible a un amplio rango de compuestos químicos, de fácil manejo en el laboratorio, y con un cultivo relativamente sencillo y barato. El objetivo del presente trabajo fue evaluar la toxicidad de agentes antiparasitarios, antimicrobianos e insecticidas sobre A. franciscana para establecer la concentración prevista que no causa efectos (PNEC) sobre los organismos marinos y obtener los niveles guía para la protección de la vida acuática. Con los nauplios II de A. franciscana, dentro de las 24 h de eclosión, se procedió a realizar los bioensayos de toxicidad calculando la Concentración letal media (CL50) a 24 h y 48 h de exposición. Se observó la siguiente secuencia de mayor a menor toxicidad a 48 h de exposición para tres agentes antiparasitarios comerciales: Mebendazol >Albendazol >Metronidazol. Con relación al efecto tóxico letal de seis agentes antimicrobianos comerciales se vio la siguiente secuencia de mayor a menor toxicidad a 48 h de exposición: Triclosan >Clotrimazol >Itraconazol >Ketoconazol >Oxitetraciclina >Mimosa. El camarón salino mostró efectos de mortalidad por acción de cinco sustancias con propiedades insecticidas, encontrándose el siguiente orden de mayor a menor mortalidad a 48 h de exposición: Cipermetrina >Rotenona >Carbaryl >Canela >Malation. Las tres sustancias químicas calificadas como muy tóxicas y que presentaron los niveles guía más bajos para la protección de la vida acuática fueron Triclosan (0,72 ug·L-1), Cipermetrina (0,84 ug·L-1) y Clotrimazol (0,97 ug·L-1). Se observó que diez (71,42%) de las sustancias químicas mostraron fuerte actividad citotóxica (AU)

Artemia franciscana "brine shrimp", is sensitive to a wide range of chemical structures, and easy handling in the laboratory and with a relatively simple and inexpensive crustacean culture. The aim of this study was to evaluate the toxicity of parasiticides agent, antimicrobials agent and insecticides on A. franciscana to establish Predicted No-Effect Concentration (PNEC) on marine organisms and obtain guidance levels for the protection of aquatic life. With A. franciscana nauplii II, within 24 h of hatching, we proceeded to perform toxicity bioassays calculating the average lethal concentration (LC50) at 24 h and 48 h of exposure. The following sequence of high to low toxicity to 48 h of exposure to three commercial antiparasitic agents were observed: Mebendazole> Albendazole> Metronidazole. Regarding the lethal toxic effect of six commercial antimicrobial agents about A. franciscana, the following sequence of toxicity at 48 h of exposure was observed: Triclosan> Clotrimazole> Itraconazole> Ketoconazole> oxytetracycline> Mimosa. The brine shrimp mortality showed effects on five substances with insecticidal properties, meeting the following order from highest to lowest mortality at 48 h of exposure Cipermethrin >Rotenone >Carbaryl >Cinnamon >Malathion. The three chemicals were classified as very toxic and presented lower levels guidance for the protection of aquatic life were Triclosan (0,72 ug·L-1), Cipermetrina (0,84 ug·L-1) y Clotrimazol (0,97 ug·L-1). Ten of chemicals (71.42%) showed strong cytotoxic activity (AU)

Inhibition of MDR3 Activity in Human Hepatocytes by Drugs Associated with Liver Injury.

MDR3 dysfunction is associated with liver diseases. We report here a novel MDR3 activity assay involving in situ biosynthesis in primary hepatocytes of deuterium (d9)-labeled PC and LC-MS/MS determination of transported extracellular PC-d9. Several drugs associated with DILI such as chlorpromazine, imipramine, itraconazole, haloperidol, ketoconazole, sequinavir, clotrimazole, ritonavir, and troglitazone inhibit MDR3 activity. MDR3 inhibition may play an important role in drug-induced cholestasis and vanishing bile duct syndrome. Several lines of evidence demonstrate that the reported assay is physiologically relevant and can be used to assess the potential of chemical entities and their metabolites to modulate MDR3 activity and/or PC biosynthesis in hepatocytes.

Biomonitoring of the Genotoxic and Hepatotoxic Effects and Oxidative Stress Potentials of Itraconazole in Pregnant Rats.

BACKGROUND: Pregnant women are more susceptible to both vaginal colonization and infection by yeast. One hundred million fungal infected patients have been treated worldwide with itraconazole (Caputo, 2003. METHOD: Itraconazole was administrated orally to pregnant rats at doses of 75, 100, or 150 mg/kg during gestational days (GD) 1 to 7 or GD 8 to 14 or GD 14 to 20. The genotoxicity and hepatotoxicity of the antifungal drug itraconazole were assessed during different periods of pregnancy using different methods. RESULTS: It was found that itraconazole was a genotoxic drug for both mothers and fetuses. This finding was observed via significant elevation in the estimated comet assay parameters (percentage of fragmented DNA, tail moment, and olive moment), percentage of fragmented DNA measured by diphenylamine assay and mixed smearing and laddering of DNA fragments of liver samples. In addition, itraconazole caused significant elevation in the level of hepatic malondialdehyde and depletion in the catalase activity and glutathione level. Furthermore, itraconazole induced histopathological alterations in the hepatic tissues of both mothers and fetuses. CONCLUSION: These findings indicate that itraconazole administration at doses of 75, 100, or 150 mg/kg during pregnancy induced maternal and fetal toxicity that could be induced by the genotoxicity and the oxidative damage.

Miltefosine is active against Sporothrix brasiliensis isolates with in vitro low susceptibility to amphotericin B or itraconazole.

Sporotrichosis is a common mycosis caused by dimorphic fungi from the Sporothrix schenckii complex. In recent years, sporotrichosis incidence rates have increased in the Brazilian state of Rio de Janeiro, where Sporothrix brasiliensis is the species more frequently isolated from patients. The standard antifungals itraconazole and amphotericin B are recommended as first-line therapy for cutaneous/lymphocutaneous and disseminated sporotrichosis, respectively, although decreased sensitivity to these drugs in vitro was reported for clinical isolates of S. brasiliensis. Here, we evaluated the activity of the phospholipid analogue miltefosine - already in clinical use against leishmaniasis - towards the pathogenic yeast form of S. brasiliensis isolates with low sensitivity to itraconazole or amphotericin B in vitro. Miltefosine had fungicidal activity, with minimum inhibitory concentration (MIC) values of 1-2 µg ml(-1). Miltefosine exposure led to loss of plasma membrane integrity, and transmission electron microscopy (TEM) analysis revealed a decrease in cytoplasmic electron density, alterations in the thickness of cell wall layers and accumulation of an electron-dense material in the cell wall. Flow cytometry analysis using an anti-melanin antibody revealed an increase in cell wall melanin in yeasts treated with miltefosine, when compared with control cells. The cytotoxicity of miltefosine was comparable to those of amphotericin B, but miltefosine showed a higher selectivity index towards the fungus. Our results suggest that miltefosine could be an effective alternative for the treatment of S. brasiliensis sporotrichosis, when standard treatment fails. Nevertheless, in vivo studies are required to confirm the antifungal potential of miltefosine for the treatment of sporotrichosis.

Does fungicide application in vineyards induce resistance to medical azoles in Aspergillus species?

This study assessed if the use of sterol demethylase inhibitor fungicides in vineyard production can induce resistance to azoles in Aspergillus strains and if it can induce selection of resistant species. We also tried to identify the Aspergillus species most prevalent in the vineyards. Two vineyards from northern Portugal were selected from "Vinhos Verdes" and "Douro" regions. The vineyards were divided into plots that were treated or not with penconazole (PEN). In each vineyard, air, soil, and plant samples were collected at three different times. The strains of Aspergillus spp. were isolated and identified by morphological and molecular techniques. We identified 46 Aspergillus section Nigri, eight Aspergillus fumigatus, seven Aspergillus lentulus, four Aspergillus wentii, two Aspergillus flavus, two Aspergillus terreus, one Aspergillus calidoustus, one Aspergillus westerdijkiae, one Aspergillus tamarii, and one Eurotium amstelodami. Aspergillus strains were evaluated for their susceptibility to medical azoles used in human therapy (itraconazole, posaconazole, and voriconazole) and to agricultural azoles (PEN) used in the prevention and treatment of plant diseases. The isolates showed moderate susceptibility to voriconazole. We did not observe any decrease of susceptibility to the medical azoles tested throughout the testing period in any of the treated plots, although some of the resistant species were isolated from there.

In utero exposure to itraconazole during different gestational periods of rats.

The present study aimed to investigate the teratogenic and genotoxic effects of itraconazole administered orally to pregnant rats on gestation days 1-7 (implantation), 8-14 (organogenesis) and 14-20 (fetal developmental period) at doses 75, 100 or 150 mg/kg b.wt. The results indicated that itraconazole had embryolethal effect when administered at a dose of 150 mg/kg b.wt throughout implantation and organogenesis periods as well as at 100 mg/kg b.wt during implantation period. Itraconazole elevated the teratogenicity when administrated at a dose of 100 mg/kg b.wt during organogenesis period, the most prominent abnormalities were abdominal hernia, protruding tongue, exencephaly, incompletely ossified, unossified or missing skull bones (mostly frontal, parietal and interparietal), abnormal vertebrae and fused and supernumerary ribs. However, minimal adverse effects were observed at doses given during the fetal developmental period. Itraconazole increased DNA damage of fetal osteocytes via significant increase in the measured comet parameters in all the treated groups, indicating that itraconazole severely affects fetal genetic material.

Inhalable highly concentrated itraconazole nanosuspension for the treatment of bronchopulmonary aspergillosis.

Cystic fibrosis (CF) patients are suffering from multiple often chronic endobronchial infection. The stiff mucus in these patients represents a compartment, which cannot easily be reached by systemic treatment. While bacterial infections are now successfully treated with repeated inhalation of antibiotics such as tobramycine, 57% of CF patients are colonized by Aspergillus species. About 10-20% of colonized patients develop symptoms of allergic bronchopulmonary aspergillosis (ABPA). While current standard of treatment of ABPA in CF patients is to suppress the allergy related symptoms by administration of glucocorticoids, itraconazole (ITRA), administered orally at high doses, can alleviate the symptoms of ABPA. However, no inhalable formulation of ITRA is available to enable local treatment of aspergillosis. The aim of this study was to describe an aqueous nanosuspension of ITRA and to characterize the pharmacokinetics after single dose inhalation. Using wet-milling with organic milling beads, a stable nanosuspension with particle size in the range of 200nm and an ITRA concentration of 20% (v/w) could be obtained, using polysorbate 80 at a concentration of 14% relative to ITRA. The suspension was stable if stored at 8°C for 3 months without particle growth and could be nebulized using standard nebulizer technologies including mesh technology and pressured air nebulizers. A 10% suspension was well tolerated upon repeated dose inhalation once daily for 7 days at a predicted dose of 45mg/kg in rats. A single dose inhalation at a predicted dose of 22.5mg/kg resulted in maximum lung tissue concentration of 21.4µg/g tissue with a terminal half-life of 25.4h. Serum concentrations were lower, with a maximum concentration of 104ng/ml at 4h after dosing and a terminal half-life of 10.5h. The data indicate that ITRA nanosuspension represents an interesting formulation for inhaled administration in CF patients suffering from ABPA. High and long lasting lung tissue concentrations well above the minimal inhibitory concentration of Aspergillus species enable once daily administration with minimal systemic exposure.

Development of a novel synergistic thermosensitive gel for vaginal candidiasis: an in vitro, in vivo evaluation.

The singular aim of the proposed work is the development of a synergistic thermosensitive gel for vaginal application in subjects prone to recurrent vaginal candidiasis and other microbial infections. The dual loading of Itraconazole and tea tree oil in a single formulation seems promising as it would elaborate the microbial coverage. Despite being low solubility of Itraconazole in tea tree oil, a homogeneous, transparent and stable solution of both was created by co-solvency using chloroform. Complete removal of chloroform was authenticated by GC-MS and the oil solution was used in the development of nanoemulsion which was further translated into a gel bearing thermosensitive properties. In vitro analyses (MTT assay, viscosity measurement, mucoadhesion, ex vivo permeation, etc.) and in vivo studies (bioadhesion, irritation potential and fungal clearance kinetics in rat model) of final formulation were carried out to establish its potential for further clinical evaluation.

In vitro antifungal activity and toxicity of itraconazole in DMSA-PLGA nanoparticles.

Itraconazole (ITZ) is a drug used to treat various fungal infections and may cause side effects. The aim of this study was to develop and evaluate the in vitro activity of DMSA-PLGA nanoparticles loaded with ITZ against Paracoccidioides brasiliensis, as well as their cytotoxicity. Nanoparticles were prepared using the emulsification-evaporation technique and characterized by their encapsulation efficiency, morphology (TEM), size (Nanosight) and charge (zeta potential). Antifungal efficacy in P. brasiliensis was determined by minimal inhibition concentration (MIC), and cytotoxicity using MTT assay. ITZ was effectively incorporated in the PLGA-DMSA nanoparticles with a loading efficiency of 72.8 +/- 3.50%. The shape was round with a solid polymeric structure, and a size distribution of 174 +/- 86 nm (Average +/- SD). The particles were negatively charged. ITZ-NANO presented antifungal inhibition (MIC = 6.25 ug/mL) against P. brasiliensis and showed lower in vitro cytotoxicity than free drug (ITZ).

Itraconazole-induced cholestasis: involvement of the inhibition of bile canalicular phospholipid translocator MDR3/ABCB4.

Biliary secretion of bile acids and phospholipids, both of which are essential components of biliary micelles, are mediated by the bile salt export pump (BSEP/ABCB11) and multidrug resistance 3 P-glycoprotein (MDR3/ABCB4), respectively, and their genetic dysfunction leads to the acquisition of severe cholestatic diseases. In the present study, we found two patients with itraconazole (ITZ)-induced cholestatic liver injury with markedly high serum ITZ concentrations. To characterize the effect of ITZ on bile formation in vivo, biliary bile acids and phospholipids were analyzed in ITZ-treated rats, and it was revealed that biliary phospholipids, rather than bile acids, were drastically reduced in the presence of clinically relevant concentrations of ITZ. Moreover, by using MDR3-expressing LLC-PK1 cells, we found that MDR3-mediated efflux of [¹4C]phosphatidylcholine was significantly reduced by ITZ. In contrast, BSEP-mediated transport of [³H]taurocholate was not significantly affected by ITZ, which is consistent with our in vivo observations. In conclusion, this study suggests the involvement of the inhibition of MDR3-mediated biliary phospholipids secretion in ITZ-induced cholestasis. Our approach may be useful for analyzing mechanisms of drug-induced cholestasis and evaluating the cholestatic potential of clinically used drugs and drug candidates.

Nanosuspension formulation of itraconazole eliminates the negative inotropic effect of SPORANOX in dogs.

Previously, it was observed that a nanosuspension formulation of itraconazole was more efficacious and yet less acutely toxic in rats as compared with the conventional solution formulation, SPORANOX (itraconazole) Injection. The present study compares the two formulations with respect to specifically myocardial contractility in conscious dogs. Motivation for doing so is highlighted by the black-box warning in the package insert for SPORANOX (itraconazole) Injection, which warns of negative inotropic effects. Conscious dogs, instrumented with a high-fidelity pressure transducer in the left ventricle, were placed in a sling for dosing and cardiac monitoring. Test and control articles were administered intravenously via a peripheral vein, and left ventricular parameters were measured continuously through 60 min from the start of dosing. As expected, SPORANOX (itraconazole) Injection caused a significant reduction in myocardial contractility as determined by the contractility index. In contrast, the itraconazole nanosuspension administered at twice the dose and at twice the rate of infusion did not result in significant changes in myocardial contractility. A novel formulation technology applied to itraconazole completely prevented the negative inotropic effect observed in conscious dogs as compared with SPORANOX (itraconazole) Injection.

Efficacy and safety of intravenous itraconazole as empirical antifungal therapy for persistent fever in neutropenic patients with hematological malignancies in Japan.

Recently, empirical antifungal therapy with intravenous itraconazole (ITCZ) for neutropenic patients with antibiotics-resistant fever has been approved by Japanese Ministry of Health, Labour and Welfare on the bases of previous multicenter trials of foreign countries. In this study, we conducted a single-arm, multicenter, prospective study in order to evaluate the efficacy of empirical ITCZ injection on Japanese patients. Sixty-eight patients with hematological diseases who underwent anticancer chemotherapy or stem cell transplantation were enrolled. In this study, we found that the overall clinical response rate to ITCZ injection was 67.6% and success rate of achieving composite endpoints including survival, defervescence during neutropenia, no breakthrough fungal infections, and no premature discontinuation of drug was 50.0%. Mild adverse reactions were observed in 6 patients (8.8%). Further analysis revealed that possible/probable deep fungal infection according to the 2002 and 2008 criteria defined by EORTC/MSG were found in 19.1 and 7.5% of the patients, respectively. Interestingly, response rate to ITCZ injection of possible/probable cases according to the 2002 and 2008 criteria was 61.5% (8/13) and 100% (5/5), respectively. These results not only proved the good efficacy and safety of empirical ITCZ injection for Japanese patients, but also indicated a utility of the drug on future "presumptive" approach.

Development and evaluation of novel itraconazole-loaded intravenous nanoparticles.

The purpose of this study was to present novel intravenous itraconazole-loaded nanoparticles (ITZ-NPs) using human serum albumin (HSA) as drug carrier materials. The ITZ-NPs were prepared by nanoparticle albumin bound technology involving a series of homogenization and lyophilization procedures. The ITZ-NPs powder could be easily reconstituted and provide stable solutions at a wide range of concentrations at 25 degrees C for 24h. In safety test, the ITZ-NPs caused mild hemolysis below the concentration of 10mg/mL and were well tolerated at the dose of 160 mg/kg in mice, indicating better biocompatibility than cyclodextrin formulation of itraconazole (ITZ-CD). The pharmacokinetic parameters of itraconazole and its major metabolite, hydroxyl-itraconazole, of ITZ-NPs had no differences from those of ITZ-CD in mice. For the ITZ-NPs group, the distributions of itraconazole in the lung, liver and spleen were higher than those for ITZ-CD group. It was of significance that ITZ-NPs increased the drug distribution in lung which was always the portal to fungal infection. These results indicate that the ITZ-NPs can be a potential intravenous formulation of itraconazole.

Imidazole antifungals, but not triazole antifungals, increase membrane Zn2+ permeability in rat thymocytes Possible contribution to their cytotoxicity.

The use of zinc as a nutritional supplement has become common in many countries. Since zinc has diverse actions, it may be difficult to predict its synergistic and/or antagonistic action in simultaneous presence of drug(s). The combination of imidazole antifungals, but not triazole antifungals, with 3-30 microM ZnCl2 significantly increased the lethality of rat thymocytes. Since intracellular Zn2+ exerts various actions on the process of cell death, there is a possibility that imidazole antifungals, but not triazole antifungals, increases concentration of intracellular Zn2+ ([Zn2+]i). To test the possibility, we examined the effects of imidazole and triazole antifungals on [Zn2+]i of rat thymocytes in absence and presence of extracellular Zn2+ by the use of FluoZin-3, a fluorescent Zn2+ indicator. Imidazole antifungals (clotrimazole, econazole, and oxiconazole) increased the [Zn2+]i in the presence of extracellular Zn2+ while it was not the case for triazole antifungals (itraconazole and fluoconazole). Thus, it is suggested that imidazole antifungals increase the membrane permeability of Zn2+. The potency order in the augmentation of FluoZin-3 fluorescence by imidazole antifungals in the presence of extracellular Zn2+ was the same as that in their cytotoxic action. Therefore, the cytotoxic action of imidazole antifungals may be related to their action on membrane Zn2+ permeability.

Itraconazole IV nanosuspension enhances efficacy through altered pharmacokinetics in the rat.

The goal of this research was to evaluate an intravenous itraconazole nanosuspension dosage form, relative to a solution formulation, in the rat. Itraconazole was formulated as a nanosuspension by a tandem process of microcrystallization followed by homogenization. Acute toxicity, pharmacokinetics, and distribution were studied in the rat, and compared with a solution formulation of itraconazole. Efficacy was studied in an immunocompromised rat model, challenged with a lethal dose of either itraconazole-sensitive or itraconazole-resistant C. albicans. Itraconazole nanosuspension was tolerated at significantly higher doses compared with a solution formulation. Pharmacokinetics of the nanosuspension were altered relative to the solution formulation. C(max) was reduced and t(1/2) was much prolonged. This occurred due to distribution of the nanosuspension to organs of the monocyte phagocytic system (MPS), followed by sustained release from this IV depot. The higher dosing of the drug, enabled in the case of the nanosuspension, led to higher kidney drug levels and reduced colony counts. Survival was also shown to be superior relative to the solution formulation. Thus, formulation of itraconazole as a nanosuspension enhances efficacy of this antifungal agent relative to a solution formulation, because of altered pharmacokinetics, leading to increased tolerability, permitting higher dosing and resultant tissue drug levels.

Murine airway histology and intracellular uptake of inhaled amorphous itraconazole.

Aerosolization of amorphous itraconazole may be a safe and effective method of pulmonary delivery. Our objective was to evaluate the histologic effects, immunogenic potential, and cellular uptake of aerosolized amorphous itraconazole. Mice received amorphous itraconazole (30mg/kg), excipient placebo, or saline control by nebulization every 12h for up to 12 days. Broncho-alveolar lavage (BAL) and formalin fixation of both lungs were conducted. BAL supernatant was assayed for IL-12 by ELISA, and cellular components were analyzed by high performance liquid chromatography-mass spectroscopy. Coronal sections of the entire lung were stained, viewed by light microscopy, and the Cimolai histopathologic inflammatory score was obtained for each lobe. No evidence of bronchiolar, peribronchiolar or perivascular inflammation was found in any treatment group, nor were epithelial ulceration or repair observed. The Cimolai histopathologic scores for amorphous itraconazole, excipient, and saline control on days 3 and 8 did not differ between groups. ELISA analysis showed no cytokine induction of IL-12. Itraconazole was detected within cells collected from BAL fluid on days 1, 3, 8 and 12. Aerosolized administration of amorphous itraconazole or excipients does not cause inflammation or changes in pulmonary histology and are not associated with pro-inflammatory cytokine production.