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.

Management of androgenic alopecia: a systematic review of the literature.

We aimed to determine the efficacy of the various available oral, topical, and procedural treatment options for hair loss in individuals with androgenic alopecia. Using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, a systematic review of the National Library of Medicine was performed. Overall, 141 unique studies met our inclusion criteria. We demonstrate that many over the counter (e.g. topical minoxidil, supplements, low-level light treatment), prescription (e.g. oral minoxidil, finasteride, dutasteride), and procedural (e.g. platelet-rich plasma, fractionated lasers, hair transplantation) treatments successfully promote hair growth, highlighting the superiority of a multifaceted and individualized approach to management.

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.

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.

Activity and safety of KEES - an oral multi-drug chemo-hormonal metronomic combination regimen in metastatic castration-resistant prostate cancer.

BACKGROUND: Metastatic castration-resistant prostate cancer (mCRPC) remains a therapeutic challenge and evidence for late-line treatments in real-life is limited. The present study investigates the efficacy and safety of an oral metronomic chemo-hormonal regimen including cyclophosphamide, etoposide, estramustine, ketoconazole and prednisolone (KEES) administered in a consecutive biweekly schedule. METHODS: A retrospective cohort study in two Swedish regions was conducted. Overall (OS) and progression-free survival (PFS), biochemical response rate (bRR) and toxicities were analyzed. RESULTS: One hundred and twenty-three patients treated with KEES after initial treatment with at least a taxane or an androgen-receptor targeting agents (ARTA) were identified. Of those, 95 (77%) had received both agents and were the primary analysis population. Median (95% CI) OS and PFS in the pre-treated population were 12.3 (10.1-15.0) and 4.4 (3.8-5.5) months, respectively. Biochemical response, defined as ≥ 50% prostate-specific antigen (PSA) reduction, occurred in 26 patients (29%), and any PSA reduction in 59 (65%). PFS was independent of prior treatments used, and KEES seemed to be effective in late treatment lines. The bRR was higher compared to historical data of metronomic treatments in docetaxel and ARTA pre-treated populations. In multivariable analyses, performance status (PS) ≥ 2 and increasing alkaline phosphatase (ALP) predicted for worse OS. Nausea, fatigue, thromboembolic events and bone marrow suppression were the predominant toxicities. CONCLUSIONS: KEES demonstrated meaningful efficacy in heavily pre-treated CRPC patients, especially those with PS 0-1 and lower baseline ALP, and had an acceptable toxicity profile.

Integrating Forward and Reverse Translation in PBPK Modeling to Predict Food Effect on Oral Absorption of Weakly Basic Drugs.

INTRODUCTION: Ketoconazole and posaconazole are two weakly basic broad-spectrum antifungals classified as Biopharmaceutics Classification System class II drugs, indicating that they are highly permeable, but exhibit poor solubility. As a result, oral bioavailability and clinical efficacy can be impacted by the formulation performance in the gastrointestinal system. In this work, we have leveraged in vitro biopharmaceutics and clinical data available in the literature to build physiologically based pharmacokinetic (PBPK) models for ketoconazole and posaconazole, to determine the suitability of forward in vitro-in vivo translation for characterization of in vivo drug precipitation, and to predict food effect. METHODS: A stepwise modeling approach was utilized to derive key parameters related to absorption, such as drug solubility, dissolution, and precipitation kinetics from in vitro data. These parameters were then integrated into PBPK models for the simulation of ketoconazole and posaconazole plasma concentrations in the fasted and fed states. RESULTS: Forward in vitro-in vivo translation of intestinal precipitation kinetics for both model drugs resulted in poor predictions of PK profiles. Therefore, a reverse translation approach was applied, based on limited fitting of precipitation-related parameters to clinical data. Subsequent simulations for ketoconazole and posaconazole demonstrated that fasted and fed state PK profiles for both drugs were adequately recapitulated. CONCLUSION: The two examples presented in this paper show how middle-out modeling approaches can be used to predict the magnitude and direction of food effects provided the model is verified on fasted state PK data.

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.

In Vitro Combination of Terbinafine with Ketoconazole Against Aspergillus Species with Terbinafine High MIC Values Isolated From Otomycosis.

Otomycosis is a common mycotic infection of the external auditory canal, and Aspergillus species are one of the most frequent causative agents worldwide. The limited antifungal arsenal, the high toxicity and side effects of antifungal agents, and the growing resistance to the currently available antifungals underscore the need for new therapeutic strategies. The present study aimed to evaluate the combined in vitro efficacy of terbinafine and ketoconazole against Aspergillus species with terbinafine high MIC values isolated from patients with otomycosis.84 Aspergillus species with high MIC values to terbinafine (≥ 4 µg/ml), consisting of A. flavus, A. tubingensis, A. niger, and A. terreus, were included in this study. The checkerboard microdilution method evaluated the in vitro interactions using the CLSI reference technique. Synergistic effects were observed for 66.67% (56/84) of all isolates (FICI ranging from 0.19 to 0.5). However, the interactions of terbinafine and ketoconazole exhibited indifference in 33.33% (28/84) of the isolates, and no antagonism was observed for any combination. The interaction of terbinafine and ketoconazole showed synergistic activity against Aspergillus species with high MIC values, suggesting that this is an alternative and promising approach for treating otomycosis.

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.

HSPiP and QbD Program-Based Analytical Method Development and Validation to Quantify Ketoconazole in Dermatokinetic Study.

Ketoconazole (KTZ) is the most potential azole anti-mycotic drug. The quantification of KTZ from various layers of the skin after topical application of lipidic nanocarriers is critical. We addressed a sensitive, specific, simple, rapid, reproducible, and economic analytical method to quantify KTZ from the treated skin homogenate using the Hansen solubility parameter (HSP, HSPiP software)-based modeling and experimental design. The software provided various HSP values for KTZ and solvents to compose the mobile phase. The Taguchi model identified the significant sets of factors to develop a robust bioanalytical method with reduced variability. In the optimization, acetonitrile (ACN) concentration (X1 as A) and the pH of mobile phase (X2 as B) were two factors against two responses (Y1: peak area and Y2: retention time). The HPLC (high-performance liquid chromatography) method validation was carried out based on US-FDA guidelines for the developed KTZ formulations (suspension, solid nanoparticles, and commercial product) extracted from the treated rat skin. The experimental solubility of KTZ was found to be maximum in the two solvents (ACN and ethyl acetate), based on HSP values. Surface response methodology (SRM) identified remarkable impact of ACN concentration and the mobile phase pH on the peak area and retention time. Analytical limits (0.17 and 0.50 µg/mL) were established for KTZ-SLNs (extracted from the skin). The method was implemented with high reproducibility, accuracy, and selectivity to quantify KTZ from the treated rat skin.

Stepwise binding of inhibitors to human cytochrome P450 17A1 and rapid kinetics of inhibition of androgen biosynthesis.

Cytochrome P450 (P450) 17A1 catalyzes the 17α-hydroxylation of progesterone and pregnenolone as well as the subsequent lyase cleavage of both products to generate androgens. However, the selective inhibition of the lyase reactions, particularly with 17α-hydroxy pregnenolone, remains a challenge for the treatment of prostate cancer. Here, we considered the mechanisms of inhibition of drugs that have been developed to inhibit P450 17A1, including ketoconazole, seviteronel, orteronel, and abiraterone, the only approved inhibitor used for prostate cancer therapy, as well as clotrimazole, known to inhibit P450 17A1. All five compounds bound to P450 17A1 in a multistep process, as observed spectrally, over a period of 10 to 30 s. However, no lags were observed for the onset of inhibition in rapid-quench experiments with any of these five compounds. Furthermore, the addition of substrate to inhibitor-P450 17A1 complexes led to an immediate formation of product, without a lag that could be attributed to conformational changes. Although abiraterone has been previously described as showing slow-onset inhibition (t1/2 = 30 min), we observed rapid and strong inhibition. These results are in contrast to inhibitors of P450 3A4, an enzyme with a larger active site in which complete inhibition is not observed with ketoconazole and clotrimazole until the changes are completed. Overall, our results indicate that both P450 17A1 reactions-17α-hydroxylation and lyase activity-are inhibited by the initial binding of any of these inhibitors, even though subsequent conformational changes occur.

Distinct effects of form selective cytochrome P450 inhibitors on cytochrome P450-mediated monooxygenase and hydrogen peroxide generating NADPH oxidase.

A characteristic of cytochrome P450 (CYP) enzymes is their ability to generate H2O2, either directly or indirectly via superoxide anion, a reaction referred to as "NADPH oxidase" activity. H2O2 production by CYPs can lead to the accumulation of cytotoxic reactive oxygen species which can compromise cellular functioning and contribute to tissue injury. Herein we determined if form selective CYP inhibitors could distinguish between the activities of the monooxygenase and NADPH oxidase activities of rat recombinant CYP1A2, CYP2E1, CYP3A1 and CYP3A2 and CYP1A1/2-enriched ß-naphthoflavone-induced rat liver microsomes, CYP2E1-enriched isoniazide-induced rat liver microsomes and CYP3A subfamily-enriched dexamethasone-induced rat liver microsomes. In the presence of 7,8-benzoflavone (2.0 µM) for CYP1A2 and 4-methylpyrazole (32 µM) or DMSO (16 mM) for CYP2E1, monooxygenase activity was blocked without affecting NADPH oxidase activity for both the recombinant enzymes and microsomal preparations. Ketoconazole (1.0 µM), a form selective inhibitor for CYP3A subfamily enzymes, completely inhibited monooxygenase activity of rat recombinant CYP3A1/3A2 and CYP3A subfamily in rat liver microsomes; it also partially inhibited NADPH oxidase activity. 7,8-benzoflavone is a type I ligand, which competes with substrate binding, while 4-methylpyrazole and DMSO are type II heme binding ligands. Interactions of heme with these type II ligands was not sufficient to interfere with oxygen activation, which is required for NADPH oxidase activity. Ketoconazole, a type II ligand known to bind multiple sites on CYP3A subfamily enzymes in close proximity to heme, also interfered, at least in part, with oxygen activation. These data indicate that form specific inhibitors can be used to distinguish between monooxygenase reactions and H2O2 generating NADPH oxidase of CYP1A2 and CYP2E1. Mechanisms by which ketoconazole inhibits CYP3A NADPH oxidase remain to be determined.

Cushing's disease: role of preoperative and primary medical therapy.

Transsphenoidal surgery is the first-line treatment for Cushing's disease. However, some situations may require the use of a primary medical treatment, such as in patients with severe life-threatening hypercortisolism, a situation which can be handled by fast-acting steroidogenesis inhibitors, instead of classical bilateral adrenalectomy. Primary medical treatment could also be considered in patients with non-severe hypercortisolism, but the evidence is far less convincing for its systematic use. The aim of this short review is to explain briefly the different circumstances in which primary medical therapy could be considered, the limits of this approach, and the way in which to initiate and monitor the treatment.

Cushing's disease: adrenal steroidogenesis inhibitors.

Cushing's disease (CD), caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor, is the most common form of Cushing's syndrome (CS), accounting for approximately 70% of cases. CD requires a prompt diagnosis, an adequate treatment selection, and long-term management to limit hypercortisolism duration and long-term complications and improve patient outcomes. Pituitary surgery is the first-line option, which is non-curative in one third of patients, therefore requiring additional treatments. Medical therapy has recently acquired an emerging role, with the availability of several drugs with different therapeutic targets, efficacy and safety profiles. The current review focuses on efficacy and safety of steroidogenesis inhibitors, and particularly the historical drugs, ketoconazole and metyrapone, and the novel drugs levoketoconazole and osilodrostat, which seem to offer a rapid, sustained, and effective disease control. Ketoconazole should be preferred in females and in patients without severe liver disease; levoketoconazole may offer an alternative to classical ketoconazole, appearing characterized by a higher potency and potential lower hepatotoxicity compared to ketoconazole. Metyrapone should be preferred in males and in patients without severe or uncontrolled hypokalemia. Both ketoconazole and metyrapone may be preferred for short-term more than for long-term treatment. Osilodrostat may represent the best choice for long-term treatment, in patients with poor compliance to the multiple daily administration schedule, and in patients without severe or uncontrolled hypokalemia. Steroidogenesis inhibitors may be used alone or in combination, and associated with pituitary directed drugs, to improve the efficacy of the single drugs, allowing a potential use of lower doses for each drug, and hypothetically reducing the rate of adverse events associated with the single drugs. Clinicians may tailor medical therapy on the specific clinical scenario, considering disease history together with patients' characteristics and hypercortisolism's degree, addressing the needs of each patient in order to improve the therapeutic outcome and to reduce the burden of illness, particularly in patients with persistent or recurrent CD.

In vitro selection of ketoconazole-pentamidine-resistant Leishmania (Viannia) braziliensis strains.

The use of ketoconazole (KTZ) plus pentamidine (PMD) could be an interesting treatment option for New World cutaneous leishmaniasis. The aim of this work was to generate KTZ- and PMD-resistant strains and to determine some characteristics of the selection process and the resulting parasites. Resistance to one or two drugs was selected on promastigotes by progressively increasing drug concentrations for eleven months. The resistance levels (IC50) to one or two drugs (synergism assay) were determined using a colorimetric resazurin methodology. The stability of the resistance phenotype (without drug pressure or after mouse passage), cross resistance with paromomycin and miltefosine, and resistance transference to intracellular amastigotes were determined. In addition, some parasite attributes compared with WT, such as growth kinetics, amastigogenesis, THP-1 cells, and mouse infection, were determined. Promastigotes resistant to KTZ or PMD were obtained three times earlier than the combined KTZ + PMD-resistant strains. Resistant parasites (promastigotes and intracellular amastigotes) were three to twelve times less susceptible to KTZ and PMD than WT parasites. The resistance phenotype on parasites was unstable, and no cross resistance was observed. Similar parasite fitness related to our evaluated characteristics was observed except for in vivo infection, where a delay of the onset of cutaneous lesions was observed after KTZ + PMD-resistant parasite infection. CONCLUSION: Combined treatment with KTZ and PMD delayed the onset of parasite resistance and was more effective in vitro than each drug separately for WT and all resistant strains. Parasites resistant to KTZ and PMD acquired similar in vitro behaviour to WT parasites, were less virulent to mice and maintained their resistance phenotype on intracellular amastigotes but not without drug pressure or after mouse infection.

Evaluation of Anti-Candida albicans Activity and Release of Ketoconazole in PMMA-G-PEG 4000 Films.

Modified release systems depend on the selection of an appropriate agent capable of controlling the release of the drug, sustaining the therapeutic action over time, and/or releasing the drug at the level of a particular tissue or target organ. Polyethylene glycol 4000 (PEG 4000) is commonly employed in drug release formulations while polymethyl methacrylate (PMMA) is non-toxic and has a good solubility in organic solvents. This study aimed at the incorporation of ketoconazole in PMMA-g-PEG 4000 and its derivatives, thus evaluating its release profile and anti-Candida albicans and cytotoxic activities. Ketoconazole was characterized and incorporated into the copolymers. The ketoconazole incorporated in the copolymer and its derivatives showed an immediate release profile. All copolymers with ketoconazole showed activity against Candida albicans and were non-toxic to human cells in the entire concentration tested.

Ketoconazole Reverses Imatinib Resistance in Human Chronic Myelogenous Leukemia K562 Cells.

Chronic myeloid leukemia (CML) is a hematologic disorder characterized by the oncogene BCR-ABL1, which encodes an oncoprotein with tyrosine kinase activity. Imatinib, a BCR-ABL1 tyrosine kinase inhibitor, performs exceptionally well with minimal toxicity in CML chemotherapy. According to clinical trials, however, 20-30% of CML patients develop resistance to imatinib. Although the best studied resistance mechanisms are BCR-ABL1-dependent, P-glycoprotein (P-gp, a drug efflux transporter) may also contribute significantly. This study aimed to establish an imatinib-resistant human CML cell line, evaluate the role of P-gp in drug resistance, and assess the capacity of ketoconazole to reverse resistance by inhibiting P-gp. The following parameters were determined in both cell lines: cell viability (as the IC50) after exposure to imatinib and imatinib + ketoconazole, P-gp expression (by Western blot and immunofluorescence), the intracellular accumulation of a P-gp substrate (doxorubicin) by flow cytometry, and the percentage of apoptosis (by the Annexin method). In the highly resistant CML cell line obtained, P-gp was overexpressed, and the level of intracellular doxorubicin was low, representing high P-gp activity. Imatinib plus a non-toxic concentration of ketoconazole (10 µM) overcame drug resistance, inhibited P-gp overexpression and its efflux function, increased the intracellular accumulation of doxorubicin, and favored greater apoptosis of CML cells. P-gp contributes substantially to imatinib resistance in CML cells. Ketoconazole reversed CML cell resistance to imatinib by targeting P-gp-related pathways. The repurposing of ketoconazole for CML treatment will likely help patients resistant to imatinib.

Synergistic Activity of Ketoconazole and Miconazole with Prochloraz in Inducing Oxidative Stress, GSH Depletion, Mitochondrial Dysfunction, and Apoptosis in Mouse Sertoli TM4 Cells.

Triazole and imidazole fungicides represent an emerging class of pollutants with endocrine-disrupting properties. Concerning mammalian reproduction, a possible causative role of antifungal compounds in inducing toxicity has been reported, although currently, there is little evidence about potential cooperative toxic effects. Toxicant-induced oxidative stress (OS) may be an important mechanism potentially involved in male reproductive dysfunction. Thus, to clarify the molecular mechanism underlying the effects of azoles on male reproduction, the individual and combined potential of fluconazole (FCZ), prochloraz (PCZ), miconazole (MCZ), and ketoconazole (KCZ) in triggering in vitro toxicity, redox status alterations, and OS in mouse TM4 Sertoli cells (SCs) was investigated. In the present study, we demonstrate that KCZ and MCZ, alone or in synergistic combination with PCZ, strongly impair SC functions, and this event is, at least in part, ascribed to OS. In particular, azoles-induced cytotoxicity is associated with growth inhibitory effects, G0/G1 cell cycle arrest, mitochondrial dysfunction, reactive oxygen species (ROS) generation, imbalance of the superoxide dismutase (SOD) specific activity, glutathione (GSH) depletion, and apoptosis. N-acetylcysteine (NAC) inhibits ROS accumulation and rescues SCs from azole-induced apoptosis. PCZ alone exhibits only cytostatic and pro-oxidant properties, while FCZ, either individually or in combination, shows no cytotoxic effects up to 320 µM.

Checkerboard Analysis To Evaluate Synergistic Combinations of Existing Antifungal Drugs and Propylene Glycol Monocaprylate in Isolates from Recalcitrant Tinea Corporis and Cruris Patients Harboring Squalene Epoxidase Gene Mutation.

Recalcitrant dermatophytic infections of the glabrous skin (tinea corporis/cruris/faciei) pose a huge challenge to health care systems. Combinations of oral and topical drugs may potentially improve cure rates, but the same has never been objectively assessed for this condition in laboratory or clinical studies. The present study was undertaken with the aim of identifying synergistic combinations of oral and topical antifungals by testing clinical isolates obtained from patients with recalcitrant tinea corporis/cruris. Forty-two patients with tinea corporis/cruris who had failed oral antifungals or had relapsed within 4 weeks of apparent clinical cure were recruited. Twenty-one isolates were identified by sequencing (all belonging to the Trichophyton mentagrophytes/T. interdigitale species complex) and subjected to antifungal susceptibility testing (AFST) and squalene epoxidase (SQLE) gene mutation analysis. Finally, five isolates, four with underlying SQLE gene mutations and one wild-type strain, were chosen for checkerboard studies using various combinations of antifungal agents. Most isolates (n = 16) showed high MICs of terbinafine (TRB) (0.5 to >16 µg/ml), with SQLE gene mutations being present in all isolates with MICs of ≥0.5 µg/ml. Synergistic interactions were noted with combinations of itraconazole with luliconazole, TRB, and ketoconazole and propylene glycol monocaprylate (PGMC) with luliconazole and with the triple combination of PGMC with luliconazole and ketoconazole. In vitro synergistic interactions provide a sound scientific basis for the possible clinical use of antifungal combinations. Hence, these synergistic combinations may be tested for clinical utility in the wake of rising resistance among dermatophytic infections of the glabrous skin.

The Influence of the Strength of Drug-Polymer Interactions on the Dissolution of Amorphous Solid Dispersions.

In an earlier report, ionic interactions between ketoconazole (KTZ), a weakly basic drug, and poly(acrylic acid) (PAA), an anionic polymer, resulted in a dramatic decrease in molecular mobility as well as reduced crystallization propensity of amorphous solid dispersion (ASD) in the solid state. On the other hand, weaker dipole-dipole interactions between KTZ and polyvinylpyrrolidone (PVP) resulted in ASDs with higher crystallization propensity (Mistry Mol Pharm., 2015, 12 (9), 3339-3350). In this work, we investigated the behavior of the ketoconazole (KTZ) solid dispersions in aqueous media. In vitro dissolution tests showed that the PAA ASD maintained the level of supersaturation for a longer duration than the PVP ASD at low polymer contents (4-20% w/w polymer). Additionally, the PAA ASDs were more resistant to drug crystallization in aqueous medium when measured with synchrotron X-ray diffractometry. Two-dimensional 1H nuclear Overhauser effect spectroscopy (NOESY) NMR cross peaks between ketoconazole and PAA confirmed the existence of drug-polymer interactions in D2O. The interaction was accompanied by a reduced drug diffusivity as monitored by 2D diffusion ordered spectroscopy (DOSY) NMR and enthalpy-driven when characterized by isothermal titration calorimetry (ITC). On the other hand, drug-polymer interactions were not detected between ketoconazole and PVP in aqueous solution, with NOESY, DOSY, or ITC. The results suggest that interactions that stabilize ASDs in the solid state can also be relevant and important in sustaining supersaturation in solution.