Randomly Methylated ß-Cyclodextrin Inclusion Complex with Ketoconazole: Preparation, Characterization, and Improvement of Pharmacological Profiles.

As a powerful imidazole antifungal drug, ketoconazole's low solubility (0.017 mg/mL), together with its odor and irritation, limited its clinical applications. The inclusion complex of ketoconazole with randomly methylated ß-cyclodextrin was prepared by using an aqueous solution method after cyclodextrin selection through phase solubility studies, complexation methods, and condition selection through single factor and orthogonal strategies. The complex was confirmed by FTIR (Fourier-transform infrared spectroscopy), DSC (differential scanning calorimetry), TGA (thermogravimetric analysis), SEM (scanning electron microscope images), and NMR (Nuclear magnetic resonance) studies. Through complexation, the water solubility of ketoconazole in the complex was increased 17,000 times compared with that of ketoconazole alone, which is the best result so far for the ketoconazole water solubility study. In in vitro pharmacokinetic studies, ketoconazole in the complex can be 100% released in 75 min, and in in vivo pharmacokinetic studies in dogs, through the complexation, the Cmax was increased from 7.56 µg/mL to 13.58 µg/mL, and the AUC0~72 was increased from 22.69 µgh/mL to 50.19 µgh/mL, indicating that this ketoconazole complex can be used as a more efficient potential new anti-fungal drug.

Preparation, characterization, and evaluation of ketoconazole-loaded pineapple cellulose green nanofiber gel.

The present study involves the isolation of cellulose nanofibers from pineapple crown waste by a combined alkali-acid treatment method. The extracted pineapple nanofibers were characterized by Fourier-transform infrared spectroscopy, differential scanning calorimetry, X-ray diffraction, scanning electron microscopy, nuclear magnetic resonance, high-resolution transmission electron microscopy, and dynamic light scattering. The extracted pineapple nanofibers were then incorporated in Carbopol 934P containing ketoconazole to prepare a ketoconazole-loaded pineapple nanofibrous gel. The prepared gel formulation was evaluated for viscosity, spreadability, extrudibility, pH, drug content, and texture profile analysis. The anticipated gel formulation was further evaluated by in vitro drug release (98.57 ± 0.58 %), ex vivo drug permeation, cytotoxicity, and histopathological studies. The permeation of the drug through skin determined by the ex-vivo diffusion study was found to be 38.27 % with a flux rate of 4.06 ± 0.26 µg/cm2/h. Further, the cytotoxicity study of pineapple nanofiber and ketoconazole-loaded nanofiber gel displayed no cytotoxic on healthy vero cells in the concentration range from 10 to 80 µg/ml. The histopathological analysis exhibited no signs of distress and inflammation. In conclusion, ketoconazole-loaded pineapple nanofiber gel could be considered as a promising delivery system for topical applications.

Interaction of the antifungal ketoconazole and its diphenylphosphine derivatives with lipid bilayers: Insights into their antifungal action.

Ketoconazole (Ke) is an important antifungal drug, and two of its diphenylphosphinemethyl derivatives (KeP: Ph2PCH2-Ke and KeOP: Ph2P(O)CH2-Ke) have shown improved antifungal activity, namely against a yeast strain lacking ergosterol, suggesting alternative modes of action for azole compounds. In this context, the interactions of these compounds with a model of the cell membrane were investigated, using POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) large unilamellar vesicles and taking advantage of the intrinsic fluorescence of Ke, KeP and KeOP. Steady-state fluorescence spectra and anisotropy, including partition and aggregation studies, as well as fluorescence lifetime measurements, were carried out. In addition, the ability of the compounds to increase membrane permeability was assessed through carboxyfluorescein leakage. The membrane/water mole fraction partition coefficients (Kp,x): (3.31 ± 0.36) x105, (8.31 ± 1.60) x105 and (4.66 ± 0.72) x106, for Ke, KeP and KeOP, respectively, show that all three compounds have moderate to high affinity for the lipid bilayer. Moreover, KeP, and particularly KeOP interact more efficiently with POPC bilayers than Ke, which correlates well with their in vitro antifungal activity. Furthermore, although the three compounds disturb the lipid bilayer, KeOP is the quickest and most efficient one. Hence, the higher affinity and ability to permeabilize the membrane of KeOP when compared to that of KeP, despite the higher lipophilicity of the latter, points to an important role of Ph2P(O)CH2- oxygen. Overall, this work suggests that membrane interactions are important for the antifungal activity of these azoles and should be considered in the design of new therapeutic agents.

Exploring the impact of CYP2D6 and UGT2B7 gene-drug interactions, and CYP-mediated DDI on oxycodone and oxymorphone pharmacokinetics using physiologically-based pharmacokinetic modeling and simulation.

Oxycodone is one of the most commonly used opioids to treat moderate to severe pain. It is metabolized mainly by CYP3A4 and CYP2D6, while only a small fraction of the dose is excreted unchanged into the urine. Oxymorphone, the metabolite primarily formed by CYP2D6, has a 40- to 60-fold higher mu-opioid receptor affinity than the parent compound. While CYP2D6-mediated gene-drug-interactions (GDIs) and drug-drug interactions (DDIs) are well-studied, they only account for a portion of the variability in oxycodone and oxymorphone exposure. The combined impact of CYP2D6-mediated GDIs and DDIs, CYP3A4-mediated DDIs, and UGT2B7 GDIs is not fully understood yet and hard to study in head-to-head clinical trials given the relatively large number of scenarios. Instead, we propose the use of a physiologically-based pharmacokinetic model that integrates available information on oxycodone's metabolism to characterize and predict the impact of DDIs and GDIs on the exposure of oxycodone and its major, pharmacologically-active metabolite oxymorphone. To this end, we first developed and verified a PBPK model for oxycodone and its metabolites using published clinical data. The verified model was then applied to determine the dose-exposure relationship of oxycodone and oxymorphone stratified by CYP2D6 and UGT2B7 phenotypes respectively, and administered perpetrators of CYP-based drug interactions. Our simulations demonstrate that the combination of CYP2D6 UM and a UGT2B7Y (268) mutation may lead to a 2.3-fold increase in oxymorphone exposure compared to individuals who are phenotyped as CYP2D6 NM / UGT2B7 NM. The extent of oxymorphone exposure increases up to 3.2-fold in individuals concurrently taking CYP3A4 inhibitors, such as ketoconazole. Inhibition of the CYP3A4 pathway results in a relative increase in the partial metabolic clearance of oxycodone to oxymorphone. Oxymorphone is impacted to a higher extent by GDIs and DDIs than oxycodone. We predict oxymorphone exposure to be highest in CYP2D6 UMs/UGT2B7 PMs in the presence of ketoconazole (strong CYP3A4 index inhibitor) and lowest in CYP2D6 PMs/UGT2B7 NMs in the presence of rifampicin (strong CYP3A4 index inducer) covering a 55-fold exposure range.

Vincristine Disposition and Neurotoxicity Are Unchanged in Humanized CYP3A5 Mice.

Previous studies have suggested that the incidence of vincristine-induced peripheral neuropathy (VIPN) is potentially linked with cytochrome P450 (CYP)3A5, a polymorphic enzyme that metabolizes vincristine in vitro, and with concurrent use of azole antifungals such as ketoconazole. The assumed mechanism for these interactions is through modulation of CYP3A-mediated metabolism, leading to decreased vincristine clearance and increased susceptibility to VIPN. Given the controversy surrounding the contribution of these mechanisms, we directly tested these hypotheses in genetically engineered mouse models with a deficiency of the entire murine Cyp3a locus [Cyp3a(-/-) mice] and in humanized transgenic animals with hepatic expression of functional and nonfunctional human CYP3A5 variants. Compared with wild-type mice, the systemic exposure to vincristine was increased by only 1.15-fold (95% confidence interval, 0.84-1.58) in Cyp3a(-/-) mice, suggesting that the clearance of vincristine in mice is largely independent of hepatic Cyp3a function. In line with these observations, we found that Cyp3a deficiency or pretreatment with the CYP3A inhibitors ketoconazole or nilotinib did not influence the severity and time course of VIPN and that exposure to vincristine was not substantially altered in humanized CYP3A5*3 mice or humanized CYP3A5*1 mice compared with Cyp3a(-/-) mice. Our study suggests that the contribution of CYP3A5-mediated metabolism to vincristine elimination and the associated drug-drug interaction potential is limited and that plasma levels of vincristine are unlikely to be strongly predictive of VIPN. SIGNIFICANCE STATEMENT: The current study suggests that CYP3A5 genotype status does not substantially influence vincristine disposition and neurotoxicity in translationally relevant murine models. These findings raise concerns about the causality of previously reported relationships between variant CYP3A5 genotypes or concomitant azole use with the incidence of vincristine neurotoxicity.

Physiologically Based Pharmacokinetic Modeling of the Drug-Drug Interaction Between CYP3A4 Substrate Glasdegib and Moderate CYP3A4 Inducers in Lieu of a Clinical Study.

Glasdegib (DAURISMO) is a hedgehog pathway inhibitor approved for the treatment of acute myeloid leukemia (AML). Cytochrome P450 3A4 (CYP3A4) has been identified as a major metabolism and clearance pathway for glasdegib. The role of CYP3A4 in the clearance of glasdegib has been confirmed with clinical drug-drug interaction (DDI) studies following the coadministration of glasdegib with the strong CYP3A4 inhibitor ketoconazole and the strong inducer rifampin. To evaluate potential drug interactions with CYP3A4 modulators, the coadministration of glasdegib with a moderate CYP3A4 inducer, efavirenz, was evaluated using physiologically based pharmacokinetic (PBPK) modeling using the Simcyp simulator. The glasdegib compound file was developed using measured physicochemical properties, data from human intravenous and oral pharmacokinetics, absorption, distribution, metabolism, and excretion studies, and in vitro reaction phenotyping results. The modeling assumptions, model parameters, and assignments of fractional CYP3A4 metabolism were verified using results from clinical pharmacokinetics (PK) and DDI studies with ketoconazole and rifampin. The verified glasdegib and efavirenz compound files, the latter of which was available in the Simcyp simulator, were used to estimate the potential impact of efavirenz on the PK of glasdegib. PBPK modeling predicted a glasdegib area under the concentration-time curve ratio of 0.45 and maximum plasma concentration ratio of 0.75 following coadministration with efavirenz. The PBPK results, in lieu of a formal clinical study, informed the drug label, with the recommendation to double the clinical dose of glasdegib when administered in conjunction with a moderate CYP3A4 inducer, followed by a resumption of the original dose 7 days post-discontinuation.

Ketoconazole-loading strategy to improve antifungal activity and overcome cytotoxicity on human renal proximal tubular epithelial cells.

Ketoconazole (KTZ), an antifungal agent used to treat localized or systemic fungal infections by inhibiting ergosterol synthesis, exhibits restricted efficacy within eukaryotic cells owing to its elevated toxicity and limited solubility in water. This study aims to improve the biological activity and overcome cytotoxic effects in the renal system of the hydrophobic KTZ by incorporating it into poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) utilizing biomaterial nano-engineering techniques. KTZ-loaded PLGA NPs (KTZ-NPs) were prepared by single emulsion solvent evaporation method and characterized by using dynamic light scattering (DLS), electrophoretic light scattering (ELS), Fourier transform-infrared (FT-IR) spectroscopy and scanning light microscopy (SEM). Particle size and zeta potential of KTZ-NPs were determined as 182.0 ± 3.27 nm and -27.4 ± 0.56 mV, respectively. Antifungal activity was analyzed with the time-kill and top agar dilution methods onCandida albicans(C. albicans) andAspergillus flavus(A. flavus). Both KTZ and KTZ-NPs caused a significant decrease inA. flavuscell growth; however, the same effect was only observed in time-killing analysis onC. albicans, indicating a methodological difference in the antifungal analysis. According to the top agar method, the MIC value of KTZ-NPs againstA. flavuswas 9.1µg ml-1, while the minimum inhibition concentration (MIC) value of KTZ was 18.2µg ml-1. The twofold increased antifungal activity indicates that nanoparticular drug delivery systems enhance the water solubility of hydrophobic drugs. In addition, KTZ-NPs were not cytotoxic on human renal proximal tubular epithelial cells (HRPTEpCs) at fungistatic concentration, thus reducing fungal colonization without cytotoxic on renal excretion system cells.

Antifungal susceptibility profile of yeasts isolated from the oral cavity of cats.

BACKGROUND: Microorganisms living in the oral cavity play an important role in health and disease of the host. Cats are susceptible to oral infections, and it is documented that fungi in the oral cavity could impact these infections. Antifungal resistance has been increasing in recent years. OBJECTIVES: This study was designed to identify yeast isolates from the oral cavity of healthy cats and to evaluate their antifungal susceptibility pattern. METHODS: Oral specimens were collected from 60 cats and cultured at 37°C for 10 days. Yeasts were isolated and identified. Their antifungal susceptibility pattern was determined according to CLSI M44-A. RESULTS: Three yeast genera were isolated, including Candida spp (55.5%), Rhodotorula spp (33.3%) and Hanseniaspora spp (11.1%). Antifungal susceptibility profiling showed that, apart from a dose-dependent effect of itraconazole, Hanseniaspora spp was susceptible to all seven drugs studied. The Candida species were susceptible to all drugs except ketoconazole (sensitivity 80%) and caspofungin (sensitivity 40%). In R. glutinis and R. minuta, 100% sensitivity was observed for amphotericin B, posaconazole, ketoconazole and voriconazole. CONCLUSIONS: The results suggest that, in comparison with humans and other animals, cats have a different oral mycoflora in terms of species, number and diversity. However, these isolates have similar susceptibility patterns to those seen in isolates from other animals and humans. More studies should be done to further characterize the oral mycobiota of cats and its role in oral infections.

Revisiting Supersaturation of a Biopharmaceutical Classification System IIB Drug: Evaluation via a Multi-Cup Dissolution Approach and Molecular Dynamic Simulation.

As a subclass of the biopharmaceutical classification system (BCS) class II, basic drugs (BCS IIB) exhibit pH-dependent solubility and tend to generate supersaturation in the gastrointestinal tract, leading to less qualified in vitro-in vivo correlation (IVIVC). This study aims to develop a physiologically based multi-cup dissolution approach to improve the evaluation of the supersaturation for a higher quality of IVIVC and preliminarily explores the molecular mechanism of supersaturation and precipitation of ketoconazole affected by Polyvinylpyrrolidone-vinyl acetate copolymer (PVPVA) and hydroxypropyl methyl-cellulose (HPMC). The concentration of ketoconazole in each cup of the dynamic gastrointestinal model (DGIM) was measured using fiber optical probes. Molecular interactions between ketoconazole and PVPVA or HPMC were simulated by Materials Studio. The results demonstrated that PVPVA and HPMC improved and maintained the supersaturation of ketoconazole. PVPVA exhibited superior precipitation inhibitory effect on ketoconazole molecule aggregation due to slightly stronger van der Waals forces as well as unique electrostatic forces, thereby further enhancing in vitro drug absorption, which correlated well with in vivo drug absorption. Compared with a conventional dissolution apparatus paddle method, the DGIM improved the mean prediction error through the IVIVC from 19.30% to 9.96%, reaching the qualification criteria. In conclusion, the physiologically based multi-cup dissolution approach enables improved evaluation of supersaturation in gastrointestinal transportation of BCS IIB drug ketoconazole, enabling screening screen precipitation inhibitors and achieving qualified IVIVC for drug formulation studies.

Treatment with the Topical Antimicrobial Peptide Omiganan in Mild-to-Moderate Facial Seborrheic Dermatitis versus Ketoconazole and Placebo: Results of a Randomized Controlled Proof-of-Concept Trial.

Facial seborrheic dermatitis (SD) is an inflammatory skin disease characterized by erythematous and scaly lesions on the skin with high sebaceous gland activity. The yeast Malassezia is regarded as a key pathogenic driver in this disease, but increased Staphylococcus abundances and barrier dysfunction are implicated as well. Here, we evaluated the antimicrobial peptide omiganan as a treatment for SD since it has shown both antifungal and antibacterial activity. A randomized, patient- and evaluator-blinded trial was performed comparing the four-week, twice daily topical administration of omiganan 1.75%, the comparator ketoconazole 2.00%, and placebo in patients with mild-to-moderate facial SD. Safety was monitored, and efficacy was determined by clinical scoring complemented with imaging. Microbial profiling was performed, and barrier integrity was assessed by trans-epidermal water loss and ceramide lipidomics. Omiganan was safe and well tolerated but did not result in a significant clinical improvement of SD, nor did it affect other biomarkers, compared to the placebo. Ketoconazole significantly reduced the disease severity compared to the placebo, with reduced Malassezia abundances, increased microbial diversity, restored skin barrier function, and decreased short-chain ceramide Cer[NSc34]. No significant decreases in Staphylococcus abundances were observed compared to the placebo. Omiganan is well tolerated but not efficacious in the treatment of facial SD. Previously established antimicrobial and antifungal properties of omiganan could not be demonstrated. Our multimodal characterization of the response to ketoconazole has reaffirmed previous insights into its mechanism of action.

Evaluation of the inhibitory effect of azoles on pharmacokinetics of lenvatinib in rats both in vivo and in vitro by UPLC-MS/MS.

BACKGROUND: Lenvatinib is a multitargeted tyrosine kinase inhibitor used in the treatment of a variety of solid tumors. This study aims to investigate the potential pharmacokinetic interactions between lenvatinib and various azoles (ketoconazole, voriconazole, isavuconazole and posaconazole) when orally administered to rats. METHODS: A total of 30 Sprague-Dawley rats were randomly allocated into five groups and administered 20 mg/kg of ketoconazole, voriconazole, isavuconazole and 30 mg/kg of posaconazole and 0.5% CMC-Na, through gavage for a duration of 7 days prior to the commencement of the experiment. On the final day, the rats were given 10 mg/kg of lenvatinib. The blood concentration of lenvatinib was determined using UPLC-MS-MS. In vitro lenvatinib were incubated with azoles and rat liver microsomes (RLMs) or human liver microsomes (HLMs). Molecular docking was lastly used to examine the binding strength of the enzymes and ligands with Autodock Vina. RESULTS: AUC and Cmax of lenvatinib significantly increased with each of the azoles (p < 0.05), whereas CLz/F decreased 0.83-flod, 0.41-fold (p < 0.05) and 0.72-fold (p < 0.01) in voriconazole, isavuconazole and ketoconazole in rats. The IC50 of lenvatinib with the azoles were 0.237, 1.300, 0.355 and 2.403 µM in RLMs and 0.160, 1.933, 3.622 and 1.831 µM in HLMs. Molecular docking analysis suggested that azoles exhibited a strong binding ability towards the target enzymes. CONCLUSION: It is imperative to acknowledge the potential drug-drug interactions mediated by CYP3A4 between azoles and lenvatinib, as these interactions hold significant implications for their clinical utilization.

Stimulatory and Inhibitory Effects of Steroid Hormones and Human Cytochrome P450 (CYP) 3A Inhibitors on Cortisol 6ß-Hydroxylation Catalyzed by CYP3A Subfamilies.

OBJECTIVE: The inhibitory and stimulatory effects of several compounds, including steroid hormones and azole antifungal agents, on cortisol 6ß-hydroxylation activity by cytochrome P450 (CYP) 3A4, polymorphically expressed CYP3A5, and fetal CYP3A7 were compared with those on testosterone 6ß-hydroxylation to clarify the catalytic properties of the predominant forms of the human CYP3A subfamily. METHODS: 6ß-Hydroxylation activities of cortisol and testosterone by CYP3A4, CYP3A5, and CYP3A7 in the absence or presence of dehydroepiandrosterone (DHEA), α-naphthoflavone (ANF), ketoconazole, itraconazole, and voriconazole were measured using high-performance liquid chromatography. RESULTS: Lower concentrations of DHEA and ANF increased cortisol 6ß-hydroxylation activities catalyzed by CYP3A4 but not those catalyzed by CYP3A5 and CYP3A7. The inhibition strength of azole antifungal agents against cortisol 6ß-hydroxylation catalyzed by all CYP3A subfamilies was similar to that of testosterone 6ß-hydroxylation. Although the Michaelis constant (Km) increased 2-fold in the presence of 20 µM DHEA compared to that of the control, the maximal velocity (Vmax) values gradually increased with increasing DHEA. For ANF, both Km and Vmax values increased, although the Km value decreased at 2.5 µM concentrations. Ketoconazole and itraconazole competitively inhibited cortisol 6ß-hydroxylation mediated by CYP3A4 with similar inhibition constants. CONCLUSION: The inhibitory/stimulatory pattern among CYP3A subfamily members differed between cortisol and testosterone, and CYP3A4 was found to be the most sensitive in terms of inhibition by azole antifungals among the CYP3A subfamily members investigated.

Tumour induction in BALB/c mice for imaging studies: An improved protocol.

Dealing with nude mice, which lack thymus and therefore are sensitive to unsterile conditions, needs special care and laboratory conditions. For preclinical studies, especially tumour imaging purposes, in which therapeutic properties of drugs or therapeutic compounds are not studied, mice with normal immune system can be a favourable alternative if they carry tumours of interest. In the current study, we introduce an optimized protocol for induction of human tumours in BALB/c mice for preclinical studies. Immune system of BALB/c mice was suppressed by administration of cyclosporine A (CsA), ketoconazole and cyclophosphamide. The tumours of MDA-MB-231, A-431 and U-87-MG human cancer cells were induced by subcutaneous injection of the cells to the immunosuppressed mice. Tumour size was calculated weekly. Histopathological and metastatic analyses were performed using haematoxylin and eosin staining. The combination of the three drugs was found to suppress immune system and decrease the numbers of white blood cells, including lymphocytes. At the eighth week, tumours with a dimension of approximately 1400 mm3 developed. Large atypical nuclei with scant cytoplasm were found to exist using histopathological analysis. No metastasis was observed in the tumour-bearing mice. A combination of CsA, ketoconazole and cyclophosphamide can be used to suppress the immune system in BALB/c mice and induce tumours with significant size.

Chemical Imaging of Pharmaceuticals in Biofilms for Wastewater Treatment Using Secondary Ion Mass Spectrometry.

The occurrence of pharmaceuticals in the aquatic environment is a global water quality challenge for several reasons, such as deleterious effects on ecological and human health, antibiotic resistance development, and endocrine-disrupting effects on aquatic organisms. To optimize their removal from the water cycle, understanding the processes during biological wastewater treatment is crucial. Time-of-flight secondary ion mass spectrometry imaging was successfully applied to investigate and analyze the distribution of pharmaceuticals as well as endogenous molecules in the complex biological matrix of biofilms for wastewater treatment. Several compounds and their localization were identified in the biofilm section, including citalopram, ketoconazole, ketoconazole transformation products, and sertraline. The images revealed the pharmaceuticals gathered in distinct sites of the biofilm matrix. While citalopram penetrated the biofilm deeply, sertraline remained confined in its outer layer. Both pharmaceuticals seemed to mainly colocalize with phosphocholine lipids. Ketoconazole concentrated in small areas with high signal intensity. The approach outlined here presents a powerful strategy for visualizing the chemical composition of biofilms for wastewater treatment and demonstrates its promising utility for elucidating the mechanisms behind pharmaceutical and antimicrobial removal in biological wastewater treatment.

Pharmacokinetic and Pharmacodynamic Consequences of Cytochrome P450 3A Inhibition on Mitragynine Metabolism in Rats.

Mitragynine, an opioidergic alkaloid present in Mitragyna speciosa (kratom), is metabolized by cytochrome P450 3A (CYP3A) to 7-hydroxymitragynine, a more potent opioid receptor agonist. The extent to which conversion to 7-hydroxymitragynine mediates the in vivo effects of mitragynine is unclear. The current study examined how CYP3A inhibition (ketoconazole) modifies the pharmacokinetics of mitragynine in rat liver microsomes in vitro. The study further examined how ketoconazole modifies the discriminative stimulus and antinociceptive effects of mitragynine in rats. Ketoconazole [30 mg/kg, oral gavage (o.g.)] increased systemic exposure to mitragynine (13.3 mg/kg, o.g.) by 120% and 7-hydroxymitragynine exposure by 130%. The unexpected increase in exposure to 7-hydroxymitragynine suggested that ketoconazole inhibits metabolism of both mitragynine and 7-hydroxymitragynine, a finding confirmed in rat liver microsomes. In rats discriminating 3.2 mg/kg morphine from vehicle under a fixed-ratio schedule of food delivery, ketoconazole pretreatment increased the potency of both mitragynine (4.7-fold) and 7-hydroxymitragynine (9.7-fold). Ketoconazole did not affect morphine's potency. Ketoconazole increased the antinociceptive potency of 7-hydroxymitragynine by 4.1-fold. Mitragynine (up to 56 mg/kg, i.p.) lacked antinociceptive effects both in the presence and absence of ketoconazole. These results suggest that both mitragynine and 7-hydroxymitragynine are cleared via CYP3A and that 7-hydroxymitragynine is formed as a metabolite of mitragynine by other routes. These results have implications for kratom use in combination with numerous medications and citrus juices that inhibit CYP3A. SIGNIFICANCE STATEMENT: Mitragynine is an abundant kratom alkaloid that exhibits low efficacy at the µ-opioid receptor (MOR). Its metabolite, 7-hydroxymitragynine, is also an MOR agonist but with higher affinity and efficacy than mitragynine. Our results in rats demonstrate that cytochrome P450 3A (CYP3A) inhibition can increase the systematic exposure of both mitragynine and 7-hydroxymitragynine and their potency to produce MOR-mediated behavioral effects. These data highlight potential interactions between kratom and CYP3A inhibitors, which include numerous medications and citrus juices.

Mechanism-based inactivation of cytochrome P450 3A by evodol.

Evodol is one of the furanoids isolated from the fruits of Evodia rutaecarpa that has been widely prescribed for the treatment of gastrointestinal diseases in China. The aim of this study was to investigate the inhibitory effect of evodol on CYP3A.A 30-min preincubation of evodol with human liver microsomes raised an obvious left IC50 shift, 3.9-fold for midazolam 1'-hydroxylation and 3.2-fold for testosterone 6ß-hydroxylation. Evodol inactivated CYP3A in a time-, concentration- and NADPH-dependent manner, with KI and kinact of 5.1 µM and 0.028 min-1 for midazolam 1'-hydroxylation and 3.0 µM and 0.022 min-1 for testosterone 6ß-hydroxylation.Co-incubation of ketoconazole attenuated the inactivation while the inclusion of glutathione (GSH) and catalase/superoxide dismutase displayed no such protection.cis-Butene-1, 4-dial (BDA) intermediate derived from evodol were trapped by glutathione and N-acetyl-lysine in microsomes and characterised by HR-MS spectra. The BDA intermediate was believed to play a key role in CYP3A inactivation. CYP3A4 and 2C9 were the primary enzymes contributing to the bioactivation of evodol.To sum up, for the first time evodol was characterised as a mechanism-based inactivator of CYP3A.

Perinatal exposure to the fungicide ketoconazole alters hypothalamic control of puberty in female rats.

Introduction: Estrogenic endocrine disrupting chemicals (EDCs) such as diethylstilbestrol (DES) are known to alter the timing of puberty onset and reproductive function in females. Accumulating evidence suggests that steroid synthesis inhibitors such as ketoconazole (KTZ) or phthalates may also affect female reproductive health, however their mode of action is poorly understood. Because hypothalamic activity is very sensitive to sex steroids, we aimed at determining whether and how EDCs with different mode of action can alter the hypothalamic transcriptome and GnRH release in female rats. Design: Female rats were exposed to KTZ or DES during perinatal (DES 3-6-12µg/kg.d; KTZ 3-6-12mg/kg.d), pubertal or adult periods (DES 3-12-48µg/kg.d; KTZ 3-12-48mg/kg.d). Results: Ex vivo study of GnRH pulsatility revealed that perinatal exposure to the highest doses of KTZ and DES delayed maturation of GnRH secretion before puberty, whereas pubertal or adult exposure had no effect on GnRH pulsatility. Hypothalamic transcriptome, studied by RNAsequencing in the preoptic area and in the mediobasal hypothalamus, was found to be very sensitive to perinatal exposure to all doses of KTZ before puberty with effects persisting until adulthood. Bioinformatic analysis with Ingenuity Pathway Analysis predicted "Creb signaling in Neurons" and "IGF-1 signaling" among the most downregulated pathways by all doses of KTZ and DES before puberty, and "PPARg" as a common upstream regulator driving gene expression changes. Deeper screening ofRNAseq datasets indicated that a high number of genes regulating the activity of the extrinsic GnRH pulse generator were consistently affected by all the doses of DES and KTZ before puberty. Several, including MKRN3, DNMT3 or Cbx7, showed similar alterations in expression at adulthood. Conclusion: nRH secretion and the hypothalamic transcriptome are highly sensitive to perinatal exposure to both DES and KTZ. The identified pathways should be exploredfurther to identify biomarkers for future testing strategies for EDC identification and when enhancing the current standard information requirements in regulation.

Identification of biomarkers and outcomes of endocrine disruption in human ovarian cortex using In Vitro Models.

Endocrine disrupting chemicals (EDCs) are raising concerns about adverse effects on fertility in women. However, there is a lack of information regarding mechanisms and effects in humans. Our study aims to identify mechanisms of endocrine disruption using two EDCs, diethylstilbestrol (DES) and ketoconazole (KTZ)1. Human ovarian cortical tissue obtained from Caesarean section patients was exposed to 10-9 M - 10-5 M KTZ and 10-10 M - 10-6 M DES in vitro for 6 days. Follicle survival and growth were studied via histology analysis and liquid-chromatography-mass spectrometry-based steroid quantification. RNA-sequencing was performed on COV434, KGN, and primary ovarian cells that were exposed for 24 h. Significantly lower unilaminar follicle densities were observed in DES 10-10 M group, whereas low KTZ exposure reduced secondary follicle density. KTZ 10-5 M reduced levels of pregnenolone and progesterone. RNA-sequencing revealed that 445 and 233 differentially expressed genes (false discovery rate < 0.1) altogether in DES and KTZ exposed groups. Gene set variation analysis showed that both chemicals modulated pathways that are important for folliculogenesis and steroidogenesis. We selected stearoyl-CoA desaturase (SCD) and 7-dehydrocholesterol reductase (DHCR7) for further validation. Up-regulation of both genes in response to KTZ was confirmed by qPCR and in situ RNA hybridization. Further validation with immunofluorescence focused on the expression of SCD in growing follicles in exposed ovarian tissue. In conclusion, SCD may serve as a potential novel human-relevant biomarker of EDC exposure and effects on ovaries.

Fungicidal Activity of Zinc Oxide Nanoparticles against Azole-Resistant Aspergillus flavus Isolated from Yellow and White Maize.

The risk of resistance development and adverse effects on human health and the environment has increased in the last decade. Furthermore, many antifungal agents fail to inhibit the pathogenesis of azole-resistant Aspergillus flavus. In this report, we isolated and identified azole-resistant A. flavus isolates from two sources of maize (white and yellow maize). The susceptibilities of Aspergillus flavus isolates were investigated by conventional antifungals such as Terbinfine, Fluconazole, Ketoconazole, Voricazole, Amphotericin, and Nystatin. Then zinc oxide nanoparticles associated with Chlorella vulgaris, which are synthesized by using the precipitation method, were examined against isolated fungi. The results showed that twelve species of white corn were isolated out of fifty isolates, while the number of isolates from the yellow corn source was only four. Interestingly, the following antifungals have an impact effect against azole-resistant A. flavus isolates: the inhibition zones of ketoconazole, voricazole, and terbinafine were 40 mm, 20 mm, and 12 mm, respectively, while the remaining antifungal agents have no effect. Similarly, the inhibition zones of the following antifungal agents were as follows: 41 mm for Terbinfine, 13 mm for Voricazole, and 11 mm for Ketoconazole against Aspergillus flavus that was isolated from yellow corn. The physiochemical characterization of zinc oxide nanoparticles provides evidence that ZnO-NPs associate with Chlorella vulgaris and have been fabricated by the precipitation method with a diameter of 25 nm. The zinc oxide nanoparticle was then used to isolate azole-resistant A. flavus, and the results show that ZnO-NPs have an effect on azole-resistant A. flavus isolation. The inhibition zone of zinc oxide nanoparticles against A. flavus (that was isolated from white corn) was 50 mm with an MIC of 50 mg/mL, while the inhibition zone of zinc oxide nanoparticles against Azole-resistant A. flavus isolated from yellow corn was 14 nm with an MIC of 25 mg/mL, which indicated that zinc oxide nanoparticles gave a better result against Azole-resistant A. flavus isolated from maize.

ERG11 Gene Variability and Azole Susceptibility in Malassezia pachydermatis.

Malassezia pachydermatis is part of the normal skin microbiota of various animal species but under certain circumstances becomes an opportunistic pathogen producing otitis and dermatitis. Commonly these Malassezia diseases are effectively treated using azoles. However, some cases of treatment failure have been reported. Alterations in the ERG11 gene have been associated with in vitro azole resistance in M. pachydermatis. In the present study, in vitro antifungal susceptibility of 89 different strains of M. pachydermatis isolated from different animal species and health status was studied. The susceptibility to fluconazole (FLZ), itraconazole (ITZ), ketoconazole and amphotericin B was tested by a disk diffusion method and 17 strains were also subjected to an ITZ E-test. Mueller-Hinton supplemented with 2% glucose and methylene blue was used as culture medium in both susceptibility assays. Multilocus sequence typing was performed in 30 selected strains using D1D2, ITS, CHS2 and ß-tubulin genes. Also, ERG11 gene was sequenced. The four antifungals tested were highly effective against most of the strains. Only two strains showed no inhibition zone to antifungals and a strain showed an increased MIC to ITZ. The study of the ERG11 sequences revealed a high diversity of DNA sequences and a total of 23 amino acid substitutions, from which only two have been previously described. Also, three deleterious substitutions (A302T, G459D and G461D) previously associated with azole resistance in this yeast were recovered. A correlation between certain genotypes and ERG11 mutations was observed. Some of the ERG11 mutations recovered were correlated with a reduced susceptibility to azoles.