Pathogenic Aspergillus Strains Identification and Antifungal Susceptibility Analysis of 452 Cases with Otomycosis in Jingzhou, China.

OBJECTIVE: To study the distribution of pathogenic Aspergillus strains of otomycosis in central China and the identification of their antifungal sensitivity. METHODS: We collected external ear canal secretions clinically diagnosed as otomycosis from April 2020 to January 2023 from the Department of Otolaryngology-Head and Neck Surgery in central China. The pathogenic Aspergillus strains were identified through morphological examination and sequencing. The antifungal sensitivity was performed using the broth microdilution method described in the Clinical Laboratory Standard Institute document M38-A3. RESULTS: In the 452 clinical strains isolated from the external ear canal, 284 were identified as Aspergillus terreus (62.83%), 92 as Aspergillus flavus (20.35%), 55 as Aspergillus niger (12.17%). In antifungal susceptibility tests the MIC of Aspergillus strains to bifonazole and clotrimazole was high,all the MIC90 is > 16 ug/mL. However, most Aspergillus isolates show moderate greatly against terbinafine, itraconazole and voriconazole. CONCLUSION: A. terreus is the most common pathogenic Aspergillus strain in otomycosis in central China. The selected topical antifungal drugs were bifonazole and clotrimazole; the drug resistance rate was approximately 30%. If the infection is persistent and requires systemic treatment, terbinafine and itraconazole can be used. The resistance of Aspergillus in otomycosis to voriconazole should be screened to avoid the systemic spread of infection in immunocompromised people and poor compliance with treatment. However, the pan-azole-resistant strain of Aspergillus should be monitored, particularly in high-risk patients with otomycosis.

Clotrimazole causes membrane depolarization and induces sub G0 cell cycle arrest in Leishmania donovani.

Clotrimazole is an FDA approved drug and is widely used as an antifungal agent. An extensive body of research is available about its mechanism of action on various cell types but its mode of killing of Leishmania donovani parasites is unknown. L. donovani causes Visceral Leishmaniasis which is a public health problem with limited treatment options. Its present chemotherapy is expensive, has adverse effects and is plagued with drug resistance issues. In this study we have explored the possibility of repurposing clotrimazole as an antileishmanial drug. We have assessed its efficacy on the parasites and attempted to understand its mode of action. We found that it has a half-maximal inhibitory concentration (IC50) of 35.75 ± 1.06 µM, 12.75 ± 0.35 µM and 73 ± 1.41 µM in promastigotes, intracellular amastigotes and macrophages, respectively. Clotrimazole is 5.73 times more selective for the intracellular amastigotes as compared to the mammalian cell. Effect of clotrimazole was reduced by ergosterol supplementation. It leads to impaired parasite morphology. It alters plasma membrane permeability and disrupts plasma membrane potential. Mitochondrial function is compromised as is evident from increased ROS generation, depolarized mitochondrial membrane and decreased ATP levels. Cell cycle analysis of clotrimazole treated parasites shows arrest at sub-G0 phase suggesting apoptotic mode of cell death.

G45 and V386 in Transmembrane Domains 1 and 8 Are Critical for the Activation of OATP1B3-Mediated E17ßG Uptake by Clotrimazole.

Organic anion-transporting polypeptides (OATPs) 1B1 and 1B3 are two highly homologous transport proteins. However, OATP1B1- and 1B3-mediated estradiol-17ß-glucuronide (E17ßG) uptake can be differentially affected by clotrimazole. In this study, by functional characterization on chimeric transporters and single mutants, we find that G45 in transmembrane domain 1 (TM1) and V386 in TM8 are critical for the activation of OATP1B3-mediated E17ßG uptake by clotrimazole. However, the effect of clotrimazole on the function of OATP1B3 is substrate-dependent as clotrimazole does not stimulate OATP1B3-mediated uptake of 4',5'-dibromofluorescein (DBF) and rosuvastatin. In addition, clotrimazole is not transported by OATP1B3, but it can efficiently permeate the plasma membrane due to its lipophilic properties. Homology modeling and molecular docking indicate that E17ßG binds in a substrate binding pocket of OATP1B3 through hydrogen bonding and hydrophobic interactions, among which its sterol scaffold forms hydrophobic contacts with V386. In addition, a flexible glycine residue at position 45 is essential for the activation of OATP1B3. Finally, clotrimazole is predicted to bind at an allosteric site, which mainly consists of hydrophobic residues located at the cytoplasmic halves of TMs 4, 5, 10, and 11.

Clotrimazole reverses macrophage M2 polarization by disrupting the PI3K/AKT/mTOR pathway.

Macrophages switch among different activation phenotypes according to distinct environmental stimuli, varying from pro-inflammatory (M1) to alternative (also named resolutive; M2) activation forms. M1-and M2-activated macrophages represent the two extremes of the activation spectrum involving multiple species, which vary in terms of function and the cytokines secreted. The consensus is that molecular characterization of the distinct macrophage population and the signals driving their activation will help in explaining disease etiology and formulating therapies. For instance, myeloid cells residing in the tumor microenvironment are key players in tumor progression and usually display an M2-like phenotype, which help tumor cells to evade local inflammatory processes. Therefore, these specific cells have been proposed as targets for tumor therapies by changing their activation profile. Furthermore, M2 polarized macrophages are phagocytic cells promoting tissue repair and wound healing and are therefore potential targets to treat different diseases. We have already shown that clotrimazole (CTZ) decreases tumor cell viability and thus tumor growth. The mechanism by which CTZ exerts its effects remains to be determined, but this drug is an inhibitor of the PI3K/AKT/mTOR pathway. In this study, we show that CTZ downregulated M2-activation markers in macrophages polarized to the M2 profile. This effect occurred without interfering with the expression of M1-polarized markers or pro-inflammatory cytokines and signaling. Moreover, CTZ suppressed NFkB pathway intermediates and disrupted PI3K/AKT/mTOR signaling. We concluded that CTZ reverses macrophage M2 polarization by disrupting the PI3K/AKT/mTOR pathway, which results in the suppression of NFkB induction of M2 polarization. In addition, we find that CTZ represents a promising therapeutic tool as an antitumor agent.

Antimicrobial Activity of Manganese(I) Tricarbonyl Complexes Bearing 1,2,3-Triazole Ligands.

BACKGROUND: Antimicrobial resistance is one of the most pressing health issues of our time. The increase in the number of antibiotic-resistant bacteria allied to the lack of new antibiotics has contributed to the current crisis. It has been predicted that if this situation is not dealt with, we will be facing 10 million deaths due to multidrug resistant infections per year by 2050, surpassing cancer-related deaths. This alarming scenario has refocused attention into researching alternative drugs to treat multidrug-resistant infections. AIMS: In this study, the antimicrobial activities of four manganese complexes containing 1,2,3,-triazole and clotrimazole ligands have been evaluated. It is known that azole antibiotics coordinated to manganese tricarbonyl complexes display interesting antimicrobial activities against several microbes. In this work, the effect of the introduction of 1,2,3,-triazole-derived ligands in the [Mn(CO)3(clotrimazole)] fragment has been investigated against one Gram-positive bacterium and five Gram-negative bacteria. METHODS: The initial antimicrobial activity of the above-mentioned complexes was assessed by determining the minimum inhibitory and bactericidal concentrations using the broth microdilution method. Growth curves in the presence and absence of the complexes were performed to determine the effects of these complexes on the growth of the selected bacteria. A possible impact on cellular viability was determined by conducting the MTS assay on human monocytes. RESULTS: Three of the Mn complexes investigated (4-6) had good antimicrobial activities against all the bacteria tested, with values ranging from 1.79 to 61.95 µM with minimal toxicity. CONCLUSIONS: Due to the increased problem of antibiotic resistance and a lack of new antibacterial drugs with no toxicity, these results are exciting and show that these types of complexes can be an avenue to pursue in the future.

IK Channel-Independent Effects of Clotrimazole and Senicapoc on Cancer Cells Viability and Migration.

Many studies highlighted the importance of the IK channel for the proliferation and the migration of different types of cancer cells, showing how IK blockers could slow down cancer growth. Based on these data, we wanted to characterize the effects of IK blockers on melanoma metastatic cells and to understand if such effects were exclusively IK-dependent. For this purpose, we employed two different blockers, namely clotrimazole and senicapoc, and two cell lines: metastatic melanoma WM266-4 and pancreatic cancer Panc-1, which is reported to have little or no IK expression. Clotrimazole and senicapoc induced a decrease in viability and the migration of both WM266-4 and Panc-1 cells irrespective of IK expression levels. Patch-clamp experiments on WM266-4 cells revealed Ca2+-dependent, IK-like, clotrimazole- and senicapoc-sensitive currents, which could not be detected in Panc-1 cells. Neither clotrimazole nor senicapoc altered the intracellular Ca2+ concentration. These results suggest that the effects of IK blockers on cancer cells are not strictly dependent on a robust presence of the channel in the plasma membrane, but they might be due to off-target effects on other cellular targets or to the blockade of IK channels localized in intracellular organelles.

Cross-linkable star-hyperbranched unimolecular micelles for the enhancement of the anticancer activity of clotrimazole.

Clotrimazole, a hydrophobic drug routinely used in the treatment of vaginal candidiasis, also shows antitumor activity. However, its use in chemotherapy has been unsuccessful to date due to its low solubility in aqueous media. In this work, new unimolecular micelles based on polyether star-hyperbranched carriers of clotrimazole are presented that can enhance solubility, and consequently the bioavailability, of clotrimazole in water. The amphiphilic constructs consisting of a hydrophobic poly(n-alkyl epoxide) core and hydrophilic corona of hyperbranched polyglycidol were synthesized in a three-step anionic ring-opening polymerization of epoxy monomers. The synthesis of such copolymers, however, was only possible by incorporating a linker to facilitate the elongation of the hydrophobic core with glycidol. Unimolecular micelles-clotrimazole formulations displayed significantly increased activity against human cervical cancer HeLa cells compared to the free drug, along with a weak effect on the viability of the normal dermal microvascular endothelium cells HMEC1. This selective activity of clotrimazole on cancer cells with little effect on normal cells was a result of the fact that clotrimazole targets the Warburg effect in cancer cells. Flow cytometric analysis revealed that the encapsulated clotrimazole significantly blocks the progression of the HeLa cycle in the G0/G1 phase and induces apoptosis. In addition, the ability of the synthesized amphiphilic constructs to form a dynamic hydrogel was demonstrated. Such a gel facilitates the delivery of drug-loaded single-molecule micelles to the affected area, where they can form a continuous, self-healing layer.

Clotrimazole is effective, safe and tolerable for the treatment of endometriosis and functions by downregulating inducible nitric oxide synthase and modulating oxidative stress biomarkers.

This study investigated the mechanism of action of clotrimazole (CTZ) and its adverse effects in a model of endometriosis. After autologous endometrial implantation, 18 rats were randomized into two treatment groups: 200 mg/kg CTZ or vehicle for 15 consecutive days. The lesion growth, implant size, glandular atrophy, nitric oxide (NO) serum levels, number of macrophage cells and inducible nitric oxide synthase (iNOS) immunoreactivity were significantly reduced in the CTZ group compared with the control. CTZ (p < 0.05) reduced the lipid peroxidation and protein carbonylation levels in the liver but did not alter the superoxide dismutase (SOD), glutathione (GSH) or glutathione S-transferase (GST) levels in the brain; however, the drug significantly reduced SOD activity and enhanced GST activity in the liver. These results suggest that CTZ interferes with reactive nitrogen species production by downregulating iNOS expression and thus enhances the antioxidant system to promote atrophy and regression of endometriotic lesions, without adverse effects on the brain and/or liver.

Clotrimazole Inhibits HCC Migration and Invasion by Modulating the ERK-p65 Signaling Pathway.

Purpose: Hepatocellular carcinoma (HCC), has a very high mortality rate and is the most common type of liver cancer. Clotrimazole, a traditional antifungal drug, has garnered considerable attention as a therapeutic strategy for HCC. However, its effects against the migration and invasion of HCC cells as well as the associated underlying mechanisms remain unclear. Therefore, in this study, we investigated its effects on HCC and attempted to elucidate the underlying molecular mechanisms. Methods: CCK-8 was used to investigate the inhibitory effect of clotrimazole on the proliferation of different types of HCC cells, and wound healing and transwell assays were performed to investigate its inhibitory effect on the invasion and migration of the HCC cells. Further, western blotting was employed to detect changes in the expression levels of epithelial mesenchymal transition (EMT)-related proteins, extracellular-regulated protein kinases (ERK), p-ERK, p65, and p-p65. We also used ERK activators in combination with clotrimazole to treat the HCC cell lines. Results: Clotrimazole inhibited the invasion and migration of HCC cells, and mechanistically, it exerted these anti-tumor effects via EMT by repressing ERK phosphorylation. Conclusion: These findings suggest that clotrimazole inhibits HCC metastasis by repressing EMT in an ERK dephosphorylation-dependent manner.

Synergistic Interaction of CPP2 Coupled with Thiazole Derivates Combined with Clotrimazole and Antineoplastic Drugs in Prostate and Colon Cancer Cell Lines.

Cell-penetrating peptides (CPPs) are small peptide sequences used mainly as cellular delivery agents that are able to efficiently deliver cargo into cells. Some CPPs also demonstrate intrinsic anticancer properties. Previously, our group developed a new family of CPP2-thiazole conjugates that have been shown to effectively reduce the proliferation of different cancer cells. This work aimed to combine these CPP2-thiazole conjugates with paclitaxel (PTX) and 5-fluorouracil (5-FU) in PC-3 prostate and HT-29 colon cancer cells, respectively, to evaluate the cytotoxic effects of these combinations. We also combined these CPP2-thiazole conjugates with clotrimazole (CLZ), an antifungal agent that has been shown to decrease cancer cell proliferation. Cell viability was evaluated using MTT and SRB assays. Drug interaction was quantified using the Chou-Talalay method. We determined that CPP2 did not have significant activity in these cells and demonstrate that N-terminal modification of this peptide enhanced its anticancer activity in both cell lines. Our results also showed an uneven response between cell lines to the proposed combinations. PC-3 cells were more responsive to the combination of CPP2-thiazole conjugates with CLZ than PTX and were more sensitive to these combinations than HT-29 cells. In addition, the interaction of drugs resulted in more synergism in PC-3 cells. These results suggest that N-terminal modification of CPP2 results in the enhanced anticancer activity of the peptide and demonstrates the potential of CPPs as adjuvants in cancer therapy. These results also validate that CLZ has significant anticancer activity both alone and in combination and support the strategy of drug repurposing coupled to drug combination for prostate cancer therapy.

Clotrimazole presents anticancer properties against a mouse melanoma model acting as a PI3K inhibitor and inducing repolarization of tumor-associated macrophages.

The immune system is a key component of tumorigenesis, with the latter promoting the development of cancer, its progression and metastasis. In fact, abundant infiltration of tumor-associated macrophages (TAM), which are M2-like macrophages, has been associated with a poor outcome in most types of cancers. Here, we show that lactate produced by murine melanoma B16F10 cells induces an M2-like profile in cultured macrophages. Further, we demonstrate that clotrimazole (CTZ), an off-target anti-tumor drug, abolishes lactate effects on the activation of macrophages and induces the expression of M1-like markers. We show that clotrimazole has cytotoxic effects on tumor cells by negatively modulating PI3K, which inhibits glycolytic metabolism and leads to a diminishing lactate production by these cells. These effects are more pronounced in cancer cells exposed to conditioned media of M2-polarized macrophages. Moreover, clotrimazole inhibits tumor growth in a murine model of implanted melanoma, reduces lactate content in a tumor microenvironment and decreases vascular endothelial growth factor expression. Finally, clotrimazole drastically diminishes TAM infiltration in the tumors, thereby inducing M1 polarization. Collectively, these findings identify a new antitumor mechanism of clotrimazole by modulating the tumor microenvironment (TME), particularly the activation and viability of TAM.

Modulation of lactate-lysosome axis in dendritic cells by clotrimazole potentiates antitumor immunity.

BACKGROUND: Dendritic cells (DCs) play a critical role in antitumor immunity, but the therapeutic efficacy of DC-mediated cancer vaccine remains low, partly due to unsustainable DC function in tumor antigen presentation. Thus, identifying drugs that could enhance DC-based antitumor immunity and uncovering the underlying mechanism may provide new therapeutic options for cancer immunotherapy. METHODS: In vitro antigen presentation assay was used for DC-modulating drug screening. The function of DC and T cells was measured by flow cytometry, ELISA, or qPCR. B16, MC38, CT26 tumor models and C57BL/6, Balb/c, nude, and Batf3-/- mice were used to analyze the in vivo therapy efficacy and impact on tumor immune microenvironment by clotrimazole treatment. RESULTS: By screening a group of small molecule inhibitors and the US Food and Drug Administration (FDA)-approved drugs, we identified that clotrimazole, an antifungal drug, could promote DC-mediated antigen presentation and enhance T cell response. Mechanistically, clotrimazole acted on hexokinase 2 to regulate lactate metabolic production and enhanced the lysosome pathway and Chop expression in DCs subsequently induced DC maturation and T cell activation. Importantly, in vivo clotrimazole administration induced intratumor immune infiltration and inhibited tumor growth depending on both DCs and CD8+ T cells and potentiated the antitumor efficacy of anti-PD1 antibody. CONCLUSIONS: Our findings showed that clotrimazole could trigger DC activation via the lactate-lysosome axis to promote antigen cross-presentation and could be used as a potential combination therapy approach to improving the therapeutic efficacy of anti-PD1 immunotherapy.

A New Anti-Estrogen Discovery Platform Identifies FDA-Approved Imidazole Anti-Fungal Drugs as Bioactive Compounds against ERα Expressing Breast Cancer Cells.

17ß-estradiol (E2) exerts its physiological effects through the estrogen receptor α (i.e., ERα). The E2:ERα signaling allows the regulation of cell proliferation. Indeed, E2 sustains the progression of ERα positive (ERα+) breast cancers (BCs). The presence of ERα at the BC diagnosis drives their therapeutic treatment with the endocrine therapy (ET), which restrains BC progression. Nonetheless, many patients develop metastatic BCs (MBC) for which a treatment is not available. Consequently, the actual challenge is to complement the drugs available to fight ERα+ primary and MBC. Here we exploited a novel anti-estrogen discovery platform to identify new Food and Drug Administration (FDA)-approved drugs inhibiting E2:ERα signaling to cell proliferation in cellular models of primary and MBC cells. We report that the anti-fungal drugs clotrimazole (Clo) and fenticonazole (Fenti) induce ERα degradation and prevent ERα transcriptional signaling and proliferation in cells modeling primary and metastatic BC. The anti-proliferative effects of Clo and Fenti occur also in 3D cancer models (i.e., tumor spheroids) and in a synergic manner with the CDK4/CDK6 inhibitors palbociclib and abemaciclib. Therefore, Clo and Fenti behave as "anti-estrogens"-like drugs. Remarkably, the present "anti-estrogen" discovery platform represents a valuable method to rapidly identify bioactive compounds with anti-estrogenic activity.

Comparison of the Inhibitory Effects of Clotrimazole and Ketoconazole against Human Carboxylesterase 2.

BACKGROUND: Both clotrimazole and ketoconazole have been verified to have an inhibitory effect on CYP3A4. hCE2 is an enzyme closely related to the side effects of several anti-cancer drugs. However, the interactions between hCE2, clotrimazole, and ketoconazole remain unclear. OBJECTIVE: The objective of this study was to investigate and compare the inhibition behaviors of the two antifungal agents, ketoconazole and clotrimazole, on the human liver microsome hCE2 and to explore their underlying mechanism. METHODS: The inhibitory effects were investigated in human liver microsomes (HLMs) using fluorescein diacetate (FD), N-(2-butyl-1,3-dioxo-2,3-dihydro-1H-phenalen-6-yl)-2-chloroacetamide (NCEN) and irinotecan (CPT- 11) as substrates of hCE2. RESULTS: Clotrimazole significantly inhibited the hCE2 activity, which was manifested by attenuated fluorescence when the substrates were FD and NCEN. The inhibitory effect of clotrimazole towards hCE2 was much stronger than that of ketoconazole, and the inhibitory behaviors displayed substrate-dependent inhibition. The IC50 value of clotrimazole, with CPT-11 as the substate, increased by 5 and 37 times more than that with FD and NCEN, respectively. Furthermore, the inhibitions of clotrimazole towards hCE2-mediated hydrolysis of FD, NCEN, and CPT-11 were all in competitive mode with the Ki values of 0.483 µM, 8.63 µM, and 29.0 µM, respectively. Molecular docking result of clotrimazole binding to hCE2 illustrated that clotrimazole could efficiently orient itself in the Z site cavity of hCE2. CONCLUSION: Clotrimazole displayed a strong inhibitory effect against hCE2, which might be used as a potential combined agent co-administrated with CPT-11 to alleviate the hCE2-mediated severe side effects.

Evaluating the Effect of Azole Antifungal Agents on the Stress Response and Nanomechanical Surface Properties of Ochrobactrum anthropi Aspcl2.2.

Azole antifungal molecules are broadly used as active ingredients in various products, such as pharmaceuticals and pesticides. This promotes their release into the natural environment. The detailed mechanism of their influence on the biotic components of natural ecosystems remains unexplored. Our research aimed to examine the response of Ochrobactrum anthropi AspCl2.2 to the presence of four azole antifungal agents (clotrimazole, fluconazole, climbazole, epoxiconazole). The experiments performed include analysis of the cell metabolic activity, cell membrane permeability, total glutathione level and activity of glutathione S-transferases. These studies allowed for the evaluation of the cells' oxidative stress response to the presence of azole antifungals. Moreover, changes in the nanomechanical surface properties, including adhesive and elastic features of the cells, were investigated using atomic force microscopy (AFM) and spectrophotometric methods. The results indicate that the azoles promote bacterial oxidative stress. The strongest differences were noted for the cells cultivated with fluconazole. The least toxic effect has been attributed to climbazole. AFM observations unraveled molecular details of bacterial cell texture, structure and surface nanomechanical properties. Antifungals promote the nanoscale modification of the bacterial cell wall. The results presented provided a significant insight into the strategies used by environmental bacterial cells to survive exposures to toxic azole antifungal agents.

Non-electrophilic TRPA1 agonists, menthol, carvacrol and clotrimazole, open epithelial tight junctions via TRPA1 activation.

Activation of the transient receptor potential A1 channel (TRPA1) by electrophilic agonists was reported to induce the opening of tight junctions (TJs). Because compounds that increase TJ permeability can be paracellular permeability enhancers, we investigated the effect of non-electrophilic TRPA1 activators, including food ingredients (menthol and carvacrol) and medication (clotrimazole), on epithelial permeability. We show that all three compounds induced increase of the permeability of fluorescein isothiocyanate-conjugated dextran (4 kDa) and decrease of transepithelial electrical resistance, accompanied by Ca2+ influx and cofilin activation in epithelial MDCK II monolayers. These phenotypes were attenuated by pretreatment of a TRPA1 antagonist, suggesting TRPA1-mediated opening of TJs. These results suggest that non-electrophilic TRPA1 activators with established safety can be utilized to regulate epithelial barriers.

Clotrimazole reduces endometriosis and the estrogen concentration by downregulating aromatase.

This study aimed to analyse the effects of clotrimazole (CTZ) on estrogen production pathway in endometriosis progression. Experimental endometriosis was induced by autologous transplantation in female Wistar rats, and then the rats were treated with clotrimazole (200 mg/kg) or vehicle, both orally and intraperitoneally, for 15 consecutive days. Serum estrogen levels and vaginal smear analyses were performed and ERα (estrogen receptor alpha) and CYP19 (cytochrome P450 aromatase) levels in the endometriotic lesions were analysed morphologically and immunohistochemically. The clotrimazole group presented a reduction in serum estrogen levels, which were not influenced by the estrous cycle of the animals. The expression of ERα and CYP19 in endometriotic lesions was also reduced in the clotrimazole group compared to the control group. Moreover, clotrimazole treatment decreased the size of the lesions, as confirmed by histological examination, which showed glandular atrophy for both routes of administration. These results suggest that clotrimazole interferes with the estrogen production pathway by downregulating CYP19 and, therefore, reducing serum estrogen levels. Thus, the drug decreases endometriotic lesion size and consequently disease progression.

Identification of Functional Transcriptional Binding Sites within Chicken Abcg2 Gene Promoter and Screening Its Regulators.

Breast cancer resistance protein (BCRP), an ATP-binding cassette (ABC) half transporter encoded by the Abcg2 gene, is reported to influence the pharmacokinetics of substrate drugs during clinical therapy. The aim of this study was to clarify the mechanisms that regulate the transcription of the chicken Abcg2 gene through cloning and characterization of its promoter region. Results showed that the Abcg2 gene is transcribed by a TATA-less promoter with several putative Sp1 sites upstream from two putative CpG islands. A luciferase reporter assay conducted both in chicken leghorn male hepatoma (LMH) cells and chicken primary hepatocytes mapped a basal promoter to nucleotides -110 to +30, which is responsible for the constitutive expression of Abcg2. The 5'-region upstream of the basal promoter was characterized by both positive and negative regulatory domains. Further, using the cell-based reporter gene assay combined with RT-PCR and drug accumulation analysis, we found that four xenobiotics, daidzein, clotrimazole, ivermectin, and lipopolysaccharide (LPS), influence the expression and function of BCRP through significant regulation of the Abcg2 gene promoter. Interaction sites with the Abcg2 gene promoter of these four selected regulators were clarified by progressive deletions and mutation assays. This study shed some light on the regulatory mechanisms involved in chicken Abcg2 gene expression and the results may have far-reaching significance regarding the usage and development of veterinary drugs.

Automated in vivo screen in zebrafish identifies Clotrimazole as targeting a metabolic vulnerability in a melanoma model.

Therapeutic approaches for cutaneous melanoma are flourishing, but despite promising results, there is an increasing number of reported primary or secondary resistance to the growing sets of drugs approved for therapy in the clinics. Combinatorial approaches may overcome resistance, as they may tackle specific weaknesses of melanoma cells, not sufficient on their own, but effective in combination with other therapies. The transgenic zebrafish line kita:ras develops melanoma with high frequency. At 3 dpf, transgenic kita:ras larvae show a hyperpigmentation phenotype as earliest evidence of abnormal melanocyte growth. Using this model, we performed a chemical screen based on automated detection of a reduction of melanocyte number caused by any of 1280 FDA or EMA approved drugs of the library. The analysis showed that 55 molecules were able to reduce by 60% or more the number of melanocytes per embryo. We further tested two compounds for each of the 5 classes, and a farnesyltransferase inhibitor (Lonafarnib), that inhibits an essential post-translational modification of HRAS and suppresses the hyperpigmentation phenotype. Combinations of Clotrimazole and Lonafarnib showed the most promising results in zebrafish embryos, allowing a dose reduction of both drugs. We performed validation of these observations in the metastatic human melanoma cell line A375M, and in normal human epithelial melanocytes (NHEM) in order to investigate the mechanism of action of Clotrimazole in blocking the proliferation of transformed melanocytes. Viability assay and analysis of energy metabolism in Clotrimazole treated cells show that this drug specifically affects melanoma cells in vitro and transformed melanocytes in vivo, having no effects on NHEM or wild type larvae. Similar effects were observed with another hit of the same class, Miconazole. Furthermore, we show that the effects of Clotrimazole are mediated by the inhibition of hexokinase activity, which is lethal to the abnormal metabolic profile of melanoma cells in vitro and in vivo. Thus, our study shows that the zebrafish can provide a phenotype-rich assay for fully automated screening approaches to identify drugs for synthetic lethal treatment in melanoma and suggest further testing of Clotrimazole in combinatorial treatments.

Diphenyl Diselenide and Clotrimazole Co-loaded into Eudragit® RS 100 Nanocapsules Formulation Has Superior Antioxidant Potential and Promising Anti-candida Activity

Abstract In the current study, nanocapsules (NC) formulations containing a co-load of clotrimazole (C), a highly prescribed antifungal drug, and diphenyl diselenide [(PhSe)2], an organoselenium compound with a promising scope of pharmacological actions, were prepared. Formulations were characterized as well as the potential toxicity, antioxidant action, and antifungal effect were assessed using in vitro techniques. The NCs were prepared employing Eudragit® RS 100 as polymeric wall and medium chain triglycerides or virgin coconut oil (CO) as core. All NC suspensions had pH around acid range, compound content close to theoretical value (1 mg/mL/drug), average diameter in nanometric range, positive values of zeta potential as well as high encapsulation efficacy and mucoadhesive property. Physicochemical stability was performed over a 30-day period and showed no modification in the aforementioned parameters to all samples. Preliminary screening of toxicological potential performed by the hen's egg test chorioallantoic membrane technique classified the formulations as non-irritant. The DPPH radical assay revealed that nanoencapsulated compounds had superior antioxidant action in comparison to their free forms (concentration range tested 1.0-25.0 µg/mL). Importantly, the formulation composed of CO and containing C and (PhSe)2 showed the highest antioxidant potential and was selected for further investigation regarding antifungal effect against some Candida spp strains. Results of in vitro antifungal assay demonstrated that the C and (PhSe)2 co-encapsulation had a minimum inhibitory concentration (MIC) values around 60. Thus, our study supplies additional data about advantages achieved by encapsulating active compounds.