Presence Of Itraconazole Research Articles

Physiologically Based Pharmacokinetic Modeling to Simulate CYP3A4-Mediated Drug-Drug Interactions for Pyrotinib.

Pyrotinib is a newly developed tyrosine kinase inhibitor whose in vivo clearance relies heavily on cytochrome P450 3A4 (CYP3A4) activity. Clinical trials are ongoing to explore the effects of coadministration with CYP3A4 perpetrators on pyrotinib exposure. The present study aims to utilize physiologically based pharmacokinetic (PBPK) modeling to predict CYP3A4-based drug interactions of pyrotinib. Pyrotinib PBPK model was developed in the PK-Sim® multicompartmental physiology structure. Physiochemical parameters were obtained from the literature, and clearance-related parameters were optimized by fitting clinical single-dose pharmacokinetic data. Pharmacokinetic parameters from the model output were compared with the observed data to validate the model predictive performance. Using validated CYP3A4 perpetrator models, we conducted PBPK simulations for drug interactions in a virtual population to explore the impacts of comedication with these perpetrators. The PBPK model accurately describes pyrotinib single- and multi-dose pharmacokinetics. The model also predicts dramatic exposure change of pyrotinib in the presence of itraconazole and rifampicin, though the impact of rifampicin is somewhat underestimated. According to model predictions, coadministration with typical potent or moderate CYP3A4 perpetrators increases pyrotinib concentration by over sixfold, extinguishing the possibility of dose adjustment for pyrotinib. A weak CYP3A4 inhibitor has minimal influence on pyrotinib pharmacokinetics. PBPK modeling provides valuable information to avoid irrational medication when receiving pyrotinib chemotherapy.

Itraconazole and neutrophil interactions in the immune-inflammatory response of paracoccidioidomycosis using a murine air pouch infection model

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.

Sensitive and effective electrochemical determination of butenafine in the presence of itraconazole using titanium nanoparticles-ionic liquid based nanocomposite sensor

The evaluation of the bioavailability of topically applied medications that act inside or under the skin is a challenging task. Herein, the current study describes a simple, quick, and low-cost electrochemical platform for determining butenafine hydrochloride (BTH) that is mainly prescribed as a treatment of dermatophytosis, applying titanium nanoparticles and an ionic liquid as outstanding mediators. In terms of low detection limits (61.63 nM) and extensive range of 2.21 × 10–7–13.46 × 10–5 M, the established electrochemical technique provided worthy analytical performance for butenafine hydrochloride sensing. The suggested sensor's practical applicability was effectively demonstrated in pharmaceutical preparations, actual stratum corneum samples, and simultaneous detection of butenafine hydrochloride and Itraconazole in pharmaceutical preparation for the first time. All of the experimental factors, like the pH and scan rate, have been investigated and optimized. Diffusion coefficients of butenafine hydrochloride at bare and modified sensors were calculated.

Sinusoidal Uptake Determines the Hepatic Clearance of Pevonedistat (TAK-924) as Explained by Extended Clearance Model.

Quantitative assessment of hepatic clearance (CLH) of drugs is critical to accurately predict human dose and drug-drug interaction (DDI) liabilities. This is challenging for drugs that involve complex transporter-enzyme interplay. In this study, we demonstrate this interplay in the CLH and DDI effect in the presence of CYP3A4 perpetrator for pevonedistat using both the conventional clearance model (CCM) and the extended clearance model (ECM). In vitro metabolism and hepatocyte uptake data showed that pevonedistat is actively transported into the liver via multiple uptake transporters and metabolized predominantly by CYP3A4 (88%). The active uptake clearance (CLact,inf) and passive diffusion clearance (CLdiff,inf) were 21 and 8.7 ml/min/kg, respectively. The CLact,inf was underpredicted as Empirical Scaling Factor of 13 was needed to recover the in vivo plasma clearance (CLplasma). Both CCM and ECM predicted CLplasma of pevonedistat reasonably well (predicted CLplasma of 30.8 (CCM) and 32.1 (ECM) versus observed CLplasma of 32.2 ml/min/kg). However, both systemic and liver exposures in the presence of itraconazole were well predicted by ECM but not by CCM (predicted pevonedistat plasma area under the concentration-time curve ratio (AUCR) 2.73 (CCM) and 1.23 (ECM))., The ECM prediction is in accordance with the observed clinical DDI data (observed plasma AUCR of 1.14) that showed CYP3A4 inhibition did not alter pevonedistat exposure systemically, although ECM predicted liver AUCR of 2.85. Collectively, these data indicated that the hepatic uptake is the rate-determining step in the CLH of pevonedistat and are consistent with the lack of systemic clinical DDI with itraconazole. SIGNIFICANCE STATEMENT: In this study, we successfully demonstrated that the hepatic uptake is the rate-determining step in the CLH of pevonedistat. Both the conventional and extended clearance models predict CLplasma of pevonedistat well however, only the ECM accurately predicted DDI effect in the presence of itraconazole, thus providing further evidence for the lack of DDI with CYP3A4 perpetrators for drugs that involve complex transporter-enzyme interplay as there are currently not many examples in the literature except prototypical OATP substrate drugs.

Quantitative Assessment of Levonorgestrel Binding Partner Interplay and Drug-Drug Interactions Using Physiologically Based Pharmacokinetic Modeling.

Levonorgestrel (LNG) is the active moiety in many hormonal contraceptive formulations. It is typically coformulated with ethinyl estradiol (EE) to decrease intermenstrual bleeding. Due to its widespread use and CYP3A4‐mediated metabolism, there is concern regarding drug‐drug interactions (DDIs), particularly a suboptimal LNG exposure when co‐administered with CYP3A4 inducers, potentially leading to unintended pregnancies. The goal of this analysis was to determine the impact of DDIs on the systemic exposure of LNG. To this end, we developed and verified a physiologically‐based pharmacokinetic (PBPK) model for LNG in PK‐Sim (version 8.0) accounting for the impact of EE and body mass index (BMI) on LNG’s binding to sex‐hormone binding globulin. Model parameters were optimized following intravenous and oral administration of 0.09 mg LNG. The combined LNG‐EE PBPK model was verified regarding CYP3A4‐mediated interaction by comparing to published clinical DDI study data with carbamazepine, rifampicin, and efavirenz (CYP3A4 inducers). Once verified, the model was applied to predict systemic LNG exposure in normal BMI and obese women (BMI ≥ 30 kg/m2) with and without co‐administration of itraconazole (competitive CYP3A4 inhibitor) and clarithromycin (mechanism‐based CYP3A4 inhibitor). Total and free LNG exposures, when co‐administered with EE, decreased 2‐fold in the presence of rifampin, whereas they increased 1.5‐fold in the presence of itraconazole. Although changes in total and unbound exposure were decreased in obese women compared with normal BMI women, the relative impact of DDIs on LNG exposure was similar between both groups.

A Combination of Itraconazole and Amiodarone Is Highly Effective against Trypanosoma cruzi Infection of Human Stem Cell–Derived Cardiomyocytes

Trypanosoma cruzi is the etiologic agent of Chagas disease (CD), which can result in severe cardiomyopathy. Trypanosoma cruzi is endemic to the Americas, and of particular importance in Latin America. In the United States and other non-endemic countries, rising case numbers have also been observed. The currently used drugs are benznidazole (BNZ) and nifurtimox, which have limited efficacy during chronic infection. We repurposed itraconazole (ICZ), originally an antifungal, in combination with amiodarone (AMD), an antiarrhythmic, with the goal of interfering with T. cruzi infection. Human pluripotent stem cells (hiPSCs) were differentiated into cardiomyocytes (hiPSC-CMs). Vero cells or hiPSC-CMs were infected with T. cruzi trypomastigotes of the II or I strain in the presence of ICZ and/or AMD. After 48 hours, cells were Giemsa stained, and infection and multiplication were evaluated microscopically. Trypanosoma cruzi infection and multiplication were evalutated also by electron microscopy. BNZ was used as a reference compound. Cell metabolism in the presence of test substances was assessed. Itraconazole and AMD showed strain- and dose-dependent interference with T. cruzi infection and multiplication in Vero cells or hiPSC-CMs. Combinations of ICZ and AMD were more effective against T. cruzi than the single substances, or BNZ, without affecting host cell metabolism, and better preserving host cell integrity during infection. Our in vitro data in hiPSC-CMs suggest that a combination of ICZ and AMD might serve as a treatment option for CD in patients, but that different responses due to T. cruzi strain differences have to be taken into account.

1357. A Combination of Itraconazole and Amiodarone Is Highly Effective Against Trypanosoma cruzi Infection of Human Stem Cell-Derived Cardiomyocytes

BackgroundTrypanosoma cruzi is the etiologic agent of Chagas disease, which can result in severe cardiomyopathy. Trypanosoma cruzi is endemic to the Americas, and of particular importance in Latin America. In the United States and other nonendemic countries, rising case numbers have been observed. The only drugs available so far are benznidazole and nifurtimox, which have limited efficacy during chronic infection. We repurposed itraconazole, originally an antifungal, in combination with amiodarone, an antiarrhythmic, with the goal to interfere with Tc infection. Both drugs inhibit sterol synthesis, while amiodarone also inhibits calcium metabolism of Trypanosoma cruzi.MethodsHuman pluripotent stem cells (HiPSC) were differentiated to cardiomyocytes (HiPSC-CM). Vero cells or HiPSC-CM were infected with the T. cruzi trypomastigotes Y strain in the presence of itraconazole and/or amiodarone. After 48 hours, infection and multiplication were evaluated by Giemsa stain. Benznidazole was used as a reference compound. Cell viability was verified by XTT assay.ResultsItraconazole and amiodarone showed dose-dependent interference with T. cruzi infection of Vero cells or HiPSC-CM. The combination of itraconazole and amiodarone was more potent than the single substances, or benznidazole at therapeutic concentrations, without affecting host cell metabolism. In addition to effects on infection, itraconazole, or amiodarone affected T. cruzi multiplication. Here, itraconazole/amiodarone combinations were more potent than either alone, both, in Vero cells, and HiPSC-CM.ConclusionOur in vitro data suggest that a combination of itraconazole and amiodarone might serve as an effective new treatment option for Chagas disease, particularly cardiac involvement, in human and animal patients.Disclosures All authors: No reported disclosures.

Drug-drug interaction and doping: Effect of non-prohibited drugs on the urinary excretion profile of methandienone.

The potential consequences of drug-drug interactions on the excretion profile of the anabolic androgenic steroid methandienone (17β-hydroxy-17α-methylandrosta-1,4-dien-3-one) are discussed. More specifically, we have evaluated by in vitro and in vivo experiments the effects of 7 non-prohibited drugs (fluconazole, ketoconazole, itraconazole, miconazole, fluoxetine, paroxetine, and nefazodone) on the main metabolic pathways of methandienone. These are selected among those most commonly used by the athletes. The in vitro assays were based on the use of human liver microsomes, specific recombinant enzyme isoforms of cytochrome P450 and uridine 5'-diphospho-glucuronosyl-transferase. The in vivo study was performed by analyzing urines collected after the oral administration of methandienone with and without the co-administration of ketoconazole. Methandienone and its metabolites were determined by liquid chromatography-mass spectrometry-based techniques after sample pretreatment including an enzymatic hydrolysis step (performed only for the investigation on phase II metabolism) and liquid/liquid extraction with t-butyl methyl-ether. The results from the in vitro experiments showed that the formation of the hydroxylated and dehydrogenated metabolites was significantly reduced in the presence of itraconazole, ketoconazole, miconazole and nefazodone, whereas the production of the 18-nor-hydroxylated metabolites and glucuronidation reactions was reduced significantly only in the presence of ketoconazole and miconazole. The analysis of the post-administration samples confirmed the in vitro observations, validating the hypothesis that drug-drug interaction may cause significant alterations in the metabolic profile of banned drugs, making their detection during doping control tests more challenging.

In-host microevolution of Aspergillus fumigatus: A phenotypic and genotypic analysis

In order to survive, Aspergillus fumigatus must adapt to specific niche environments. Adaptation to the human host includes modifications facilitating persistent colonisation and the development of azole resistance. The aim of this study is to advance understanding of the genetic and physiological adaptation of A. fumigatus in patients during infection and treatment. Thirteen A. fumigatus strains were isolated from a single chronic granulomatous disease patient suffering from persistent and recurrent invasive aspergillosis over a period of 2 years. All strains had identical microsatellite genotypes and were considered isogenic. Whole genome comparisons identified 248 non-synonymous single nucleotide polymorphisms. These non-synonymous mutations have potential to play a role in in-host adaptation. The first 2 strains isolated were azole susceptible, whereas later isolates were itraconazole, voriconazole and/or posaconazole resistant. Growth assays in the presence and absence of various antifungal stressors highlighted minor changes in growth rate and stress resistance, with exception of one isolate showing a significant growth defect. Poor conidiation was observed in later isolates. In certain drug resistant isolates conidiation was restored in the presence of itraconazole. Differences in virulence were observed as demonstrated in a Galleria mellonella infection model. We conclude that the microevolution of A. fumigatus in this patient has driven the emergence of both Cyp51A-independent and Cyp51A-dependent, azole resistance mechanisms, and additional phenotypes that are likely to have promoted fungal persistence.

Development of stability-enhanced ternary solid dispersions via combinations of HPMCP and Soluplus® processed by hot melt extrusion

The aim of this study was to evaluate a novel combination of hydroxypropyl methylcellulose phthalate (HPMCP-HP-50) and Soluplus® polymers for enhanced physicochemical stability and solubility of the produced amorphous solid dispersions (ASDs). This was achieved using hot melt extrusion (HME) to convert the crystalline active pharmaceutical ingredient (API) into a more soluble amorphous form within the ternary systems. Itraconazole (ITZ), a Biopharmaceutics Classification System class II (BCS II) API, was selected as the model drug. The ASDs were characterized by Powder X-Ray diffraction (PXRD), Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA), Fourier Transform Infrared (FTIR) spectroscopy, Solid State Nuclear Magnetic Resonance (ssNMR) and dissolution studies. The data showed that the ASDs were physically and chemically stable at 20°C and 50% RH over 12 months. PXRD results indicated that the ITZ in the ASDs was in the amorphous state and no recrystallization occurred. DSC scans confirmed that each formulation exhibited a single intermediate glass transition (Tg), around 96.4°C, indicating that ITZ was completely miscible in the polymeric blends of HPMCP and Soluplus® at up to 30% (w/w) drug loading and that the two polymers were miscible with each other in the presence of ITZ. The FTIR analysis indicated the formation of strong hydrogen bonding between ITZ, HPMCP and Soluplus®. The dissolution end-point of the ASDs was determined to be approximately 10 times greater than that of the crystalline ITZ.

Lipid composition of the mycelium of the fungus Mucor hiemalis cultivated with trehalose, triacylglycerols, and itraconazole

We investigated the growth and cell lipid composition of the fungus Mucor hiemalis VKMF-1431 cultivated under aerobic conditions in the presence of the morphogenetic agents itraconazole, exogenous triacylglycerols, and trehalose. The sporangiospores of a 6-day culture were used as inocula. Under these conditions, the fungus produced mycelium; nevertheless, solitary yeastlike cells also developed on the glucose-containing medium and in the presence of itraconazole and sterilized triacylglycerols (sTAGs). No yeastlike growth occurred in the system with trehalose and with unsterilized (native) TAGs (nTAGs). With trehalose and nTAGs in the cultivation medium, the ratio between PEA and PC, the two main types of membrane lipids, was low. This testified to a relatively high PC percentage and, accordingly, a stable structure and a highly functional state of the membranes. Moreover, if the development of the fungus occurred exclusively as mycelium formation, the level of polyunsaturated fatty acids (γ-linolenic and arachidonic acid) increased in the presence of trehalose and that of linoleic acid increased in the presence of nTAGs. These results may suggest that unsaturated fatty acids and membrane lipids are related to the cell wall formation and the implementation of morphogenetic programs in mucorous fungi.

Abstract 149: Antitumor effect of itraconazole on hematological malignant cells by suppression of Hedgehog signal transduction via inhibition of lipid raft formation

Abstract [Introduction and aim] Itraconazole (ITCZ) is one of the antifungal agents that inhibits fungal cell membrane ergosterol synthesis. ITCZ has been widely used for the treatment of fungal infections with high efficiency. Recently, anti-tumor effect of ITCZ was reported for solid tumors (Kim J et al. Cancer Cell 2010;17:388-399). During the treatment of hematological malignancies, ITCZ has been quite often used for invasive fungal infections; however, there has been no report for the effect of ITCZ on hematological malignancies. Therefore, we aimed to investigate the anti-tumor effect of ITCZ on hematological malignancies in the present study. [Methods] DAUDI, Jurkat, K562, Karpas299 cell lines were used as a model of hematological malignancies in the present study. At first, MTT assays were performed by treating the cells to determine if ITCZ possessed anti-tumor effect on the cell growth. Then, comprehensive analysis of intracellular signal transductions was performed utilizing reporter assay system. To evaluate the involvement of lipid raft formation by ITCZ, Gli1 expression concerning the Hedgehog signal transduction was determined by real-time PCR and western blotting, and fluorescent staining of the cells using lipid raft marker was also performed. [Results] ITCZ inhibited the cell growth by 40 to 60% determined by MTT assays in all four cell lines tested in the present study. Comprehensive analysis of intracellular signal transductions utilizing reporter assay system in Karpas299 cells showed the strong decrease of Gli1 transcription, implying the inhibition of Hedgehog signal transduction. Concerning Hedgehog signal pathway, Gli1 protein has been reported to be overexpressed in tumor cells and considered to be involved in the tumor growth, and Gli1 has also been reported to localize in lipid raft. Therefore, we hypothesized that ITCZ might influenced on the lipid raft of cell membrane and then Gli1 expression was suppressed, leading to the weakened Hedgehog signal transduction and cell growth inhibition. Fluorescent staining of Jurkat cells using lipid raft marker showed that the presence of ITCZ apparently inhibited the formation of lipid raft on cell membrane. This finding was quite similar compared to that observed when lipid raft inhibitor was added to cell culture medium. [Conclusions] ITCZ has an inhibitory effect on the cell growth in the cultured cell lines derived from hematological malignancies as same as the previous finding observed in solid tumors. Our results further indicated that ITCZ inhibited the formation of lipid raft on cell membrane, and then Hedgehog signal transduction should be weakened, finally leading to the cell growth inhibition. Taken together, the tumor inhibition by ITCZ should be favorable effect and may enhance the efficacy of the treatment of various hematological malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 149. doi:1538-7445.AM2012-149

Itraconazol Inhibits the P-Glycoprotein Pump and Increases Vincristine Induced Toxicity in Patients with Acute Lymphoblastic Leukemia Carrying a Genetic Variant of the MDR1 Gene

Abstract 231One of the most well known drug interactions in pediatric oncology concerns the interaction of vincristine, one of the cornerstones in treatment of acute lymphoblastic leukemia( ALL), and the antifungal agent itraconazole. Several studies have shown that the co-administration of itraconazole with vincristine can induce increased neurotoxicity. It is believed that this increased neurotoxicity is mainly caused by the inhibitory effect of itraconazole on the CYP3A subfamily enzymes system (e.g. CYP3A4/5) and that, depending on the genetic variation, some patients are more prone to exhibit this neurotoxicity. However, itraconazole can also inhibit the efflux pump P-glycoprotein (Pg) but so far, data on the role of the Pg pump on vincristine toxicity are lacking. We therefore performed a pharmacogenetic analysis in 103 Caucasian pediatric patients with ALL analyzing both the MDR1 gene and the CYP3A5 gene in the germ-line. CYP3A5 was not associated with vincristine-related toxicity in our cohort. However, one SNP in MDR1 (3435C>T) was significantly associated with developing peripheral neuropathy (Odds Ratio [OR] = 3.8, 95% confidence interval [CI] 1.2 – 11.9, p=0.026). It is known that this genetic variant of the MDR gene (3435T/2677T), leads to a reduced expression of the Pg-pump. Peripheral neurotoxicity in patients carrying this SNP was present even without the co-administration of itraconazole. A subgroup of these patients did receive itraconazole, and the patients with the genetic variant 3435T/2677T of MDR1 showed not only increased peripheral neurotoxicity, but also developed central nervous toxicity (OR=6.4, 95% CI 1.1 – 37.4 p=0.038) upon combined VCR-itraconazole therapy.To gain more insight in the mechanisms of increased vincristine induced toxicity during azole treatment, in vitro experiments using the LLC-PK1-MDR cell line that over-expresses the MDR1 gene and has no expression of the CYP3A4, were performed. The LLC-PK1-MDR cells were resistant to vincristine exposure in vitro, but became sensitive for VCR after inhibition of the Pg-pump bij PSC833. In contrast, LLC-PK1-MDR1 cells exposed to vincristine in the presence of itraconazole (0.5 and 5 microgram/ml) showed significantly decreased survival in a dose dependent way (LD50 at a dose of 10 −4M and 10−7 M vincristine at dose respectively of 0.5 and 5.0 microgram/ml itraconazole), in contrast to a cell survival of 100% with the same dose of vincristine itraconazole (p< 0.001).These results indicate that itraconazol indeed inhibits the efflux Pg-pump which may explain the CNS toxicity that occurred in our patient group during the period in which itraconazole was co-administerd during vincristine treatment. Based on these data, we conclude that the Pg- pump plays a dominant role in increased vincristine toxicity during azole treatment. Furthermore, those patients carrying the genetic variant 3435T/2677T of the MDR gene are at increased risk for this toxicity due to reduced expression of the efflux pump Pg. Disclosures:No relevant conflicts of interest to declare.

Influence of Itraconazole on T‐cell Immune Deviation

Objective: The objective of this study was to determine if the presence of itraconazole can alter the activation and differentiation pattern of T‐cells. Proliferation assays utilizing immunofluorescence and flow cytometry were used to determine the presence, number, and type of T‐cell subpopulations within each experimental group. Also, we sough to verify that the presence of itraconazole can alter the production of Th2 mediated cytokines. RT‐PCR was employed to measure relative numbers of gene transcripts for the various characteristic cytokines associated with T‐cell subpopulations. Design. Prospective, controlled experiment. Subjects. Samples of whole blood were be taken from healthy volunteers. Intervention(s): Following isolation of naive T cells, cells were divided into 4 groups and immune deviated in the following fashion. Controls cell lines were activated with anti‐CD3 and CD 28 and additional cytokines and antibodies were added to deviate towards Th1 and Th2 conditions. Experimental groups consisted of similar conditions with the addition of itraconazole. Results. Successful immune deviation of controls was performed. With the addition of itraconazole to Th2 deviating conditions, IL‐5, IL‐13, and IFN gamma were all noted to decrease (both by PCR and supernatant assay). Conclusions. Itraconazole inhibits the deviation of naive T cells towards the Th2 mediated pathway. METHODS and MATERIALS Samples of whole blood were taken from healthy volunteers (n = 10). Whole blood was washed and then filtered through bead column with anti‐CD45RA beads to isolate naive T cells. Naive T cells were then divided into 6 groups specified as follows: Cells were incubated in respective solutions for 96 hours. Following incubation, T cells were then harvested into 2 groups. Group 1 cells were stained and fixed, in preparation for flow cytometry to determine the number of each T‐cell population (Th1 vs Th2). Real time PCR was then performed on the group 2 cells, to determine the relative number of mRNA transcripts for IFN‐g, IL‐4, IL‐5, and IL‐13 as compared to a reference protein. Assays were then performed on incubated supernatant to evaluate for cytokine production in each of the respective 6 groups, assaying for IL‐2, IL‐4, IL‐5, IL‐9, IL‐13, INF‐g and reference proteins. INTRODUCTION Chronic rhinosinusitis (CRS) is a chronic inflammatory condition affecting the paranasal sinuses and nasal cavities which has a significant adverse impact on the quality of life and daily functioning of its sufferers.1‐3 No one specific etiology has been defined and it is more likely that this term represents either multiple different diseases with or without various stages of severity along their spectra. Certain subsets of CRS have been strongly associated with a Th2 mediated inflammatory process and, in turn, it has been theorized that at least a portion of these subsets are associated with an abnormal reaction to the presence of fungus in the nasal cavity and paranasal sinuses.4‐9 Previous work has demonstrated the ability of itraconazole to mitigate the production of Th2 associated cytokines when non‐specific T‐cell cultures are grown in its presence. Additionally, there has been some data regarding the clinical efficacy of treating CRS patients with itraconazole.10 However, no work has been done evaluating the potential role that it may have in affecting the initial differentiation process of the Th0 cell upon activation. Several studies have related anti‐mycotics to the suppression of T cell (IL‐2) and Th2‐mediated cytokines (IL‐4 and IL‐5). Bruserad demonstrated that itraconazole suppressed the cytokine‐driven proliferation of human AML cell lines.11 Kanda et al showed results that anti‐mycotics reduced the anti‐ CD3/CD28‐induced mRNA expression and promoter activities for interleukin‐4 and interleukin‐5 in atopic and normal volunteer human T‐cells.12

Antifungal Therapy with Itraconazole Impairs the Anti-Lymphoma Effects of Rituximab by Inhibiting Recruitment of CD20 to Cell Surface Lipid Rafts

Immunotherapy with rituximab alone or in conjunction with chemotherapy has significantly improved the treatment outcome of B-cell lymphoma patients. Nevertheless, a subpopulation of patients does not respond to rituximab. The reason for treatment failure as well as the exact mechanism of action is still uncertain. The function of rituximab has long been associated with the partitioning of CD20 molecules to membrane microdomains. Here, we show that concomitant antifungal treatment with itraconazole impairs the rituximab anti-lymphoma effect both in vitro and in vivo. At the molecular level, recruitment of CD20 to lipid rafts is inhibited in the presence of itraconazole. Furthermore, calcium influx, which is crucial for rituximab-mediated cell death, was nearly completely abolished by itraconazole treatment. In contrast, the antifungal drug caspofungin did not inhibit CD20 recruitment to lipid rafts, nor did it affect calcium influx or the cytotoxic effect of rituximab. The finding that itraconazole also abolished the cytotoxic effects of other therapeutic antibodies directed against lipid raft-associated molecules (i.e., CD20 and CD52) but not those against the non-raft-associated molecule CD33 further supported our proposed mechanism of action. Our results argue that concomitant medications must be adjusted carefully to achieve optimal antitumor effects with monoclonal antibodies.

Antifungal Therapy with Itraconazole Impairs Rituximab′s Anti-Lymphoma Effect by Inhibiting CD20 Recruitment to Lipid Rafts.

Abstract 2735Poster Board II-711Patients with hematologic malignancies have a high risk of developing invasive fungal infections. The higher risk is attributed to host defense impairment due to intensive cytotoxic chemotherapies, hematopoetic stem cell transplantation, and immunosuppressive agents. As early treatment initiation in patients with invasive fungal infections has a profound impact on mortality rates, different antifungal treatment strategies like prophylaxis, empirical, pre-emptive and targeted treatment after proven fungal infection have been developed. Azoles are the most broadly used antifungal drugs inhibiting CYP51. They play a pivotal role in the treatment and prophylaxis of systemic and dermal mycoses. Azoles inhibit the sterol 14a-demethylase activity and block sterol biosynthesis, which is lethal in unicellular organisms. However, in mammalian cells, it has been shown that they lower endogenous cholesterol production. As lipid rafts consist of sphingolipids and cholesterol and lipid rafts have been demonstrated to be crucial for rituximab induced cell death, we asked whether rituximab exhibits its full anti-lymphoma effect in the presence of azoles.We now demonstrate that antifungal treatment with itraconazole impairs rituximab's anti-lymphoma effect both in vitro and in vivo. Using a mouse xenograft model, no tumor growth was observed in mice treated with rituximab. In contrast, in the presence of itraconazole there was tumor growth, indicating that the concentration was sufficient to antagonize rituximab′s anti-lymphoma effect. On the molecular level, CD20 raft recruitment was inhibited in the presence of itraconazole. Furthermore, the translocation of CD20 into lipid rafts has been shown to be crucial for calcium influx and apoptosis. Therefore, we tested the rituximab induced calcium flux in the absence and presence of itraconazole. Indeed, calcium flux was almost completely abolished in the presence of itraconazole. The fact that antibody treatment failure in the presence of itraconazole only affects targeted therapy against raft associated molecules i.e. CD20 and CD52 but not the non raft associated molecule CD33 further supports our proposed mechanism of action. Together, our data suggest that concomitant treatment of itraconazole and rituximab attenuates rituximab's anti-lymphoma effect. As chemotherapy by itself has high response rates in lymphoma, the loss of rituximab mediated cell death in the presence of itraconacole may be missed in our daily routine. Disclosures:No relevant conflicts of interest to declare.

Effects of high-dose itraconazole treatment on lipoproteins in men

Epidemiological studies have convincingly demonstrated a positive association between LDL-cholesterol (LDL-C) and coronary artery disease but, in the case of HDL-C, there is an inverse association. Administration of high doses of the antifungal agent ketoconazole (800 mg/d) reduces serum concentrations of total cholesterol and LDL-C and there is a tendency for an increase in HDL-C. Our goal was to examine whether high-dose itraconazole raises HDL-C in subjects with normal levels of cholesterol. 8 male patients with onychomycosis received 2 one-week cycles of treatment with itraconazole at a dose of 400 mg once daily in an open, prospective exploratory trial. Serum levels of itraconazole and its active metabolite hydroxyitraconazole were determined using high-performance liquid chromatography at the end of each treatment cycle. Fasting levels of serum lipoproteins and triglycerides were measured twice using routine enzymatic assays at the beginning and end of each cycle. The effects of itraconazole and hydroxyitraconazole on HDL-C metabolism were assessed in vitro using a human Caco-2 cell line and analyzing apoA-I levels with an enzyme-linked immunosorbent assay. During itraconazole treatment total cholesterol and LDL-C decreased on average by 12% (p < 0.001) and 17% (p < 0.001), respectively, whereas HDL-C increased by 21% (p < 0.001). The ratio LDL: HDL-C, an index of atherogenic risk, decreased by 30% (p < 0.001). Incubation of Caco-2 cells in the presence of itraconazole and hydroxyitraconazole for 3 hours resulted in a significant increase in apoA-I concentration in the medium (913 and 412%, respectively) compared with control. In addition to its inhibitory effect on cholesterol synthesis, high-dose itraconazole (400 mg/d) causes a significant decrease in serum LDL-C and, in contrast to ketoconazole, a significant increase in HDL-C. In vitro studies with Caco-2 cells indicate that the latter observation might be caused by an increase in apoA-I levels.

MODULATION OF THE P-GLYCOPROTEIN-MEDIATED INTESTINAL SECRETION OF IVERMECTIN: IN VITRO AND IN VIVO ASSESSMENTS

The everted gut sac method was used to assess the role of the P-glycoprotein (P-gp) on the intestinal secretion of ivermectin (IVM), an antiparasitic widely used in human and veterinary medicine. The work included the evaluation of two different P-gp modulators [itraconazole (ITZ) and valspodar (PSC833)] used at equimolar doses in the rat. Furthermore, the influence of both P-gp modulator agents on the disposition kinetics of IVM in plasma, liver, and gastrointestinal tissues was characterized. For the in vitro experiments, ileal sacs were incubated with IVM (3 microM) in the presence or absence of either ITZ (10 microM) or PSC833 (10 microM). In the in vivo experiments, male Wistar rats were randomly allocated to three groups (n=18) and subcutaneously treated with IVM (200 microg/kg-1), alone and coadministered with ITZ (5 mg, two doses) or PSC833 (8.6 mg, two doses). Animals were sacrificed between 6 and 96 h. Blood, liver, and gastrointestinal samples were collected. IVM concentrations were determined by high performance liquid chromatography. The rate of IVM accumulation in the intestinal wall of everted sacs was significantly higher after its incubation with ITZ (0.115 nmol/g/min) and PSC833 (0.238 nmol/g/min) than that obtained after the incubation without the P-gp modulators (0.016 nmol/g/min). In agreement with the in vitro experiment, the presence of ITZ and PSC833 induced an enhancement in the concentrations of IVM in plasma and gastrointestinal tissues. The results obtained in the current work, both under in vivo and in vitro conditions, confirm the relevance of P-gp-mediated transport to the intestinal secretion of IVM.

Pharmacokinetics of telithromycin: application to dosing in the treatment of community-acquired respiratory tract infections

ABSTRACTIntroduction: Telithromycin is the first ketolide antibacterial approved for treating community-acquired pneumonia, acute exacerbations of chronic bronchitis, and acute bacterial sinusitis in adults. The purpose of this article is to review the main pharmacokinetic properties of telithromycin and their application to the treatment of these infections.Methods: Sources of information were identified through a Medline search (up to March 2005).Main findings: The absolute oral bioavailability of telithromycin is ≈ 57%, which is unaffected by food intake. At the recommended 800 mg once-daily oral dosing regimen, telithromycin reaches a steady-state concentration of ≈ 2 µg/mL in plasma and has an elimination half-life of ≈ 10 hours. Telithromycin shows extensive tissue distribution and penetrates effectively into bronchopulmonary tissue and epithelial lining fluid. Since elimination of telithromycin occurs via multiple pathways – the highest proportion (70%) through metabolism – impairment of a single pathway has a limited impact on telithromycin exposure. Dose adjustments are unnecessary in elderly patients or in individuals with hepatic impairment or mild to moderate renal impairment. A reduced dose could be recommended in patients with severe renal impairment. Telithromycin is metabolized primarily in the liver, approximately half of which is via the cytochrome P450 (CYP) 3A4 system. Telithromycin AUC(0–24 h) increased by 1.5- to 2.0‐fold in the presence of itraconazole and ketoconazole. Administration of telithromycin with drugs metabolized via CYP3A4 may result in increased exposure to the co-administered drug, as shown for simvastatin (5.3‐fold) and midazolam (6‐fold). Co-administration of telithromycin minimally increases (1.2- to 1.4‐fold) exposure to theophylline, digoxin, and metoprolol. Although telithromycin does not affect the pharmacokinetics of warfarin, consideration should be given to monitoring prothrombin times/INR in patients receiving telithromycin and oral anticoagulants simultaneously.Conclusion: Overall, the pharmacokinetic/pharmacodynamic properties of telithromycin indicate that this ketolide antibacterial is a valuable and convenient treatment option for community-acquired respiratory tract infections.

Effect of itraconazole on the pharmacokinetics and pharmacodynamics of fexofenadine in relation to the genetic polymorphism

Our objective was to evaluate the effect of itraconazole, a P-glycoprotein inhibitor, on the pharmacokinetics and pharmacodynamics of fexofenadine, a P-glycoprotein substrate, in relation to the multidrug resistance 1 gene (MDR1) G2677T/C3435T haplotype. A single oral dose of 180 mg fexofenadine was administered to 7 healthy subjects with the 2677GG/3435CC (G/C) haplotype and 7 with the 2677TT/3435TT (T/T) haplotype. One hour before the fexofenadine dose, either 200 mg itraconazole or placebo was administered to the subjects in a double-blinded, randomized, crossover manner with a 2-week washout period. Histamine-induced wheal and flare reactions were measured to assess the effects on the antihistamine response. In the placebo phase, pharmacokinetic parameters of fexofenadine showed no statistically significant difference between 2 MDR1 haplotypes; the area under the curve from time 0 to infinity (AUC(0-infinity)) of fexofenadine in the T/T and G/C groups was 5194.0 +/- 1910.8 and 4040.4 +/- 1832.2 ng.mL(-1).h(-1), respectively (P = .271), and the oral clearance (CL/F) was 530.9 +/- 191.1 and 806.0 +/- 355.3 mL.h(-1).kg(-1), respectively (P = .096). The disposition of itraconazole, a substrate of P-glycoprotein, was not significantly different between the 2 haplotypes. After itraconazole pretreatment, however, the differences in fexofenadine pharmacokinetics became statistically significant; the mean fexofenadine AUC(0-infinity) in the T/T group was significantly higher than that in the G/C group (15,630.6 +/- 5070.0 and 9252.9 +/- 2044.1 ng/mL.h, respectively; P = .007), and CL/F of the T/T subjects was lower than that of the G/C subjects (167.0 +/- 33.3 and 292.3 +/- 42.2 mL.h(-1).kg(-1), respectively; P < .001). Itraconazole pretreatment caused more than a 3-fold increase in the peak concentration of fexofenadine and the area under the curve to 6 hours compared with the placebo phase. This resulted in a significantly higher suppression of the histamine-induced wheal and flare reactions in the itraconazole pretreatment phase compared with those in the placebo phase. The effect of MDR1 G2677T/C3435T haplotypes on fexofenadine disposition are magnified in the presence of itraconazole. Itraconazole pretreatment significantly altered the disposition of fexofenadine and thus its peripheral antihistamine effects.