N-3-Methylbutyl-benzisoselenazol-3(2H)-one Exerts Antifungal Activity In Vitro and in a Mouse Model of Vulvovaginal Candidiasis.

In the present work, we evaluated the antifungal activities of two novel ebselen analogs, N-allyl-benzisoselenazol-3(2H)-one (N-allyl-bs) and N-3-methylbutylbenzisoselenazol-3(2H)-one (N-3mb-bs). Colorimetric and turbidity assays were performed to determine the minimum inhibitory concentration (MIC) of these compounds in S1 (fluconazole-sensitive) and S2 (fluconazole-resistant) strains of C. albicans. N-3mb-bs was more active than the N-allyl-bs compound. It is noteworthy that the concentration of N-3mb-bs observed to inhibit fungal growth by 50% (18.2 µM) was similar to the concentration observed to inhibit the activity of the yeast plasma membrane H+-ATPase (Pma1p) by 50% (19.6 µM). We next implemented a mouse model of vulvovaginal candidiasis (VVC) using the S1 strain and examined the mouse and yeast proteins present in the vaginal lavage fluid using proteomics. The yeast proteins detected were predominately glycolytic enzymes or virulence factors associated with C. albicans while the mouse proteins present in the lavage fluid included eosinophil peroxidase, desmocollin-1, and gasdermin-A. We then utilized the N-3mb-bs compound (12.5 mg/kg) in the mouse VVC model and observed that it significantly reduced the vaginal fungal burden, histopathological changes in vagina tissue, and expression of myeloperoxidase (MPO). All in all, the present work has identified a potentially promising drug candidate for VVC treatment.

Selected N-Terpenyl Organoselenium Compounds Possess Antimycotic Activity In Vitro and in a Mouse Model of Vulvovaginal Candidiasis.

In the present work, a series of N-terpenyl organoselenium compounds (CHB1-6) were evaluated for antimycotic activity by determining the minimum inhibitory concentration (MIC) for each compound in fluconazole (FLU)-sensitive (S1) and FLU-resistant (S2) strains of Candida albicans (C. albicans). The most active compounds in the MIC screen were CHB4 and CHB6, which were then evaluated for cytotoxicity in human cervical cancer cells (KB-3-1) and found to be selective for fungi. Next, CHB4 and CHB6 were investigated for skin irritation using a reconstructed 3D human epidermis and both compounds were considered safe to the epidermis. Using a mouse model of vulvovaginal candidiasis (VVC), CHB4 and CHB6 both exhibited antimycotic efficacy by reducing yeast colonization of the vaginal tract, alleviating injury to the vaginal mucosa, and decreasing the abundance of myeloperoxidase (MPO) expression in the tissue, indicating a reduced inflammatory response. In conclusion, CHB4 and CHB6 demonstrate antifungal activity in vitro and in the mouse model of VVC and represent two new promising antifungal agents.

Oral lipid nanocomplex of BRD4 PROteolysis TArgeting Chimera and vemurafenib for drug-resistant malignant melanoma.

BRAF inhibitors (BRAFi) like vemurafenib (VEM) provide initial regression in mutated melanoma but rapidly develop resistance. Molecular pathways responsible for development of resistance against VEM finally converge towards the activation of oncogenic c-Myc. We identified an epigenetic approach to inhibit the c-Myc expression and resensitize BRAFi-resistant melanoma cells. ARV-825 (ARV) was employed as a BRD4 targeted PROteolysis TArgeting Chimera that selectively degrades the BRD4 to downregulate c-Myc. ARV synergistically enhanced the cytotoxicity of VEM in vitro to overcome its resistance in melanoma. Development of ARV and VEM-loaded lipid nanocomplex (NANOVB) significantly improved their physicochemical properties for oral delivery. Most importantly, oral administration of NANOVB substantially inhibited tumor growth at rate of 41.07 mm3/day in nude athymic mice. NANOVB treatment resulted in prolonged survival with 50% of mice surviving until the experimental endpoint. Histopathological analysis revealed significant tumor necrosis and downregulation of Ki-67 and BRD4 protein in vivo. Promising in vivo antitumor activity and prolonged survival demonstrated by NANOVB signifies its clinical translational potential for BRAFi-resistant melanoma.

Nanoformulation of a novel potent ebselen analog for treatment of vulvovaginal candidiasis.

Background: Vulvovaginal candidiasis is primarily caused by Candida albicans (C. albicans). Here, a novel organoselenium compound (G20) was synthesized and evaluated for anti-Candida activity. Methods: Growth-inhibition studies and medium acidification assays to assess the inhibition of the yeast plasma membrane H+-ATPase (Pma1p) were carried out in vitro using G20. A self-nanoemulsifying formulation (SNEP) of G20 was prepared and evaluated for antimycotic activity in a mouse model. Results: G20 inhibited the growth of C. albicans through a mechanism that, at least in part, involves the inhibition of Pma1p. The G20-SNEP formulation significantly reduced vaginal colonization and vaginal inflammation relative to yeast-infected but untreated control mice. Conclusion: G20-SNEP exhibits potent antimycotic activity in a mouse model of vulvovaginal candidiasis.

Dysregulation of the mTOR pathway by mechlorethamine.

Mechlorethamine (HN2) is a derivative of the chemical warfare agent sulfur mustard (SM) and cutaneous exposure to HN2 is associated with dermal-epidermal junction (DEJ) disruption (vesication). The primary purpose of the present study was to investigate the effect of HN2 on the mammalian target of rapamycin (mTOR) signaling pathway using an in vivo mouse ear vesicant model (MEVM). To this end, the ears of male C57BL/ 6 J mice were exposed to a single topical dose of HN2 (100 mM) or vehicle control (DMSO). Mice were then euthanized 30 min, 1 h or 24 h following exposure. Mouse ear skin exposed to HN2 and biopsied 24 h thereafter exhibited increased tissue expression of Raptor, an important member of the mTORC1 complex, relative to vehicle treated samples. HN2 reduced the downstream effectors phospho S6 (Ser 240/244) ribosomal protein and phospho 4E-BP1 (Thr 37/46) of the mTOR pathway in the epidermis at 30 min, 1 h and 24 h following HN2 exposure but not in the dermis. These results support the hypothesis that HN2-mediated cutaneous toxicity involves dysregulation of the mTOR signaling pathway in the epidermis.

Combination of ebselen and hydrocortisone substantially reduces nitrogen mustard-induced cutaneous injury.

The purpose of the present study was to investigate the vesicant countermeasure effects of hydrocortisone (HC) and ebselen (EB-1), administered as monotherapy or as a combination treatment. The mouse ear vesicant model (MEVM) was utilized and test doses of HC (0.016, 0.023, 0.031, 0.047, 0.063, 0.125 or 0.250 mg/ear), EB-1 (0.125, 0.187, 0.250, 0.375 or 0.500 mg/ear) or the combination of HC + EB-1 were topically applied at 15 min, 4 h and 8 h after nitrogen mustard exposure. Ear punch biopsies were obtained 24 h after mechlorethamine (HN2) exposure. Compared to control ears, ear tissues exposed topically to HN2 (0.500 µmol/ear) presented with an increase in ear thickness, vesication, TUNEL fluorescence and expression of matrix metalloproteinase 9 (MMP-9) and inducible nitric oxide synthase (iNOS). In contrast, HN2 exposed ears treated topically with EB-1 showed a significant decrease in morphometric thickness and vesication vs. HN2 alone. Ear tissues exposed to HN2 and then treated with HC also demonstrated reductions in morphometric thickness and vesication. Combination treatment of HC + EB-1 was found to be the most effective at reducing HN2-induced ear edema and vesication. The combination also dramatically decreased HN2-mediated cutaneous expression of iNOS and MMP-9 and decreased HN2-induced TUNEL staining. Taken together, our study demonstrates that the combination of HC + EB-1 is an efficacious countermeasure to HN2.

Evaluation of the antifungal activity of an ebselen-loaded nanoemulsion in a mouse model of vulvovaginal candidiasis.

Vulvovaginal candidiasis (VVC), caused by Candida albicans, is a common infection in women affecting their quality of life. Standard antifungal drugs (e.g., fluconazole, itraconazole) are typically fungistatic or rendered ineffective due to drug resistance indicating an urgent need to build an arsenal of novel antifungal agents. To surmount this issue, we tested the hypothesis that the organoselenium compound ebselen (EB) possesses antifungal efficacy in a mouse model of VVC. EB is a poorly water-soluble drug and DMSO as a vehicle has the potential to exhibit cytotoxic effects when administered in vivo. EB loaded self-nanoemulsifying preconcentrate (EB-SNEP) was developed, characterized in vitro, and tested in a mouse model of VVC. In vivo studies carried out with EB-SNEP (12.5 mg/kg) showed a remarkable decrease in infection by ~562-fold compared to control (infected, untreated animals). Taken together, EB nanoemulsion proved to be an effective and promising antifungal agent.

Antifungal Activity of Novel Formulations Based on Terpenoid Prodrugs against C. albicans in a Mouse Model.

Carvacrol (CAR), a phenolic monoterpenoid, has been extensively investigated for its antimicrobial and antifungal activity. As a result of its poor physicochemical properties, water soluble carvacrol prodrugs (WSCPs) with improved water solubility were previously synthesized and found to possess antimicrobial activity. Here, three novel CAR analogs, WSCP1, WSCP2, and WSCP3, were tested against fluconazole (FLU)-sensitive and -resistant strains where they showed greater antifungal activity than CAR against C. albicans. The probable mechanism by which the CAR prodrugs exert the antifungal activity was studied. Results from medium acidification assays demonstrated that the CAR and its synthetically designed prodrugs inhibit the yeast plasma membrane H+-ATPase (Pma1p), an essential target in fungi. In other words, in vitro data indicated that CAR analogs can prove to be a better alternative to CAR considering their improved water solubility. In addition, CAR and WSCP1 were developed into intravaginal formulations and administered at test doses of 50 mg/kg in a mouse model of vulvovaginal candidiasis (VVC). Whereas the CAR and WSCP1 formulations both exhibited antifungal efficacy in the mouse model of VVC, the WSCP1 formulation was superior to CAR, showing a remarkable decrease in infection by ~120-fold compared to the control (infected, untreated animals). Taken together, a synthetically designed prodrug of CAR, namely WSCP1, proved to be a possible solution for poorly water-soluble drugs, an inhibitor of an essential yeast pump in vitro and an effective and promising antifungal agent in vivo.

ß-cyclodextrin polymer/Soluplus® encapsulated Ebselen ternary complex (EßpolySol) as a potential therapy for vaginal candidiasis and pre-exposure prophylactic for HIV.

Epidemiological findings have discussed recurrent and persistent vulvovaginal candidiasis to be a major manifestation of HIV infected women. Conversely, women with vulvovaginal candidiasis have higher risk of acquiring HIV transmitted during intercourse. Common treatments for such conditions include combined antiretroviral and antifungal therapy. Drug-Drug interaction is a major problem encountered due to common CYP450 metabolic pathway of azoles and antiretroviral drugs. Ebselen (EB), lipophilic, organo-selenium compound has demonstrated promising anti-HIV and anti-fungal activity. The aim of current research was to develop and characterize a rapidly soluble and non-cytotoxic vaginal film of ebselen which could serve dual purpose of treating vulvovaginal candidiasis and pre-exposure prophylactic (PrEP) against HIV. Ebselen/cyclodextrin polymer/Soluplus® (1:10:10) ternary complex (EßpolySol) showed 200 fold enhancement in aqueous solubility and no degradation of EB in thermogravimetry analysis. EßpolySol film with tensile strength of 33.12 ± 1.98 N/cm2 disintegrated within 30 sec, presented instant drug release with no apparent precipitation in simulated vaginal fluid. EßpolySol film showed compatibility with HEC-1A monolayer and HeLa cells compared to VCF®. EßpolySol film showed MIC of 20 µM against Candida species and IC50 of 0.71 µM against HIV.

Ebselen nanoemulgel for the treatment of topical fungal infection.

Superficial mycoses are the fungal infections of skin, hair and nail which affect thousands of people worldwide. Emerging resistance to azole antifungals is a common problem in the treatment of superficial or systemic fungal infection. Ebselen (EB), an organoselenium compound, has demonstrated promising activity against pathogenic yeasts. EB showed negligible dynamic and kinetic solubility in water (~ 4.2 µg/mL) which severely limits the scope of conventional formulations. The objective of present study was to develop and characterize a novel topical nanoemulgel of EB for enhancing solubility and permeability. Based on saturation solubility study, EB loaded self-nanoemulsifying preconcentrate (EB-P) was prepared using Dimethylacetamide, Kolliphor® ELP and Medium chain triglyceride which spontaneously formed 54.82 ± 1.26 nm size nanoglobules with zeta potential of -1.69 mV. Nanoemulgel was prepared by homogenous dispersion of EB-P in various gel/ointment bases. Scanning electron microscopy images showed significant drug precipitation in nanoemulgels prepared without Soluplus®. Rheological study confirmed shear thinning behavior of Soluplus® based HPMC K4M (SBH) gel. EB-P loaded SBH showed 2.3 and 5-fold higher Strat-M® deposition of EB compared to HPMC gel and Aquaphor®, respectively. EB-P showed marked anti-fungal activity at 20 µM against Candida albicans and Candida tropicalis while terbinafine was ineffective even at 100 µM concentration. Thus, topical nanoemulgel of EB could be a promising alternative to existing therapy for treatment of candidiasis.

Ebselen oxide attenuates mechlorethamine dermatotoxicity in the mouse ear vesicant model.

Mechlorethamine (HN2) is an alkylating agent and sulfur mustard mimetic. Topical exposure to HN2 is associated with tissue blistering. Previous work in our laboratory has shown that ebselen (EB-1) possesses anti-vesicant, anti-inflammatory, anti-bacterial, anti-fungal, and cytoprotective properties, both in vivo and in vitro. We recently reported that ebselen oxide (EB-2), an analog of EB-1 with a tetravalent selenium atom, also possesses anti-bacterial and anti-fungal activity and confers cytoprotection against HN2 in vitro. The purpose of the present study was to determine the vesicant countermeasure potential of EB-2 using the mouse ear vesicant model (MEVM). Compared to control ears, mouse ears exposed to a single dose of HN2 (0.500 µmol/ear) showed an increase in wet weights, ear thickness, hyperplasia, vesication, and inflammatory cell infiltration after 24 h. Fluorescence microscopy of terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL)-stained sections showed that the occurrence of apoptosis extended from the epidermis of the HN2-treated side, all the way to the contralateral epidermis. In contrast, HN2-exposed ears treated topically with EB-2 at a test dose of 0.250 mg/ear showed a significant decrease in wet weight (12% less vs. HN2 alone), morphometric thickness (13% less vs. HN2 alone), and vesication. In addition, TUNEL staining revealed that HN2 ears treated with EB-2 (0.250 mg/ear) showed a decrease in apoptosis as compared to the HN2 group. EB-2 also reduced the abundance of matrix metalloproteinase-9 (MMP-9) in ear tissues exposed to HN2. Taken together, our study demonstrates that EB-2 is an efficacious countermeasure to HN2.

Assessment of the time-dependent dermatotoxicity of mechlorethamine using the mouse ear vesicant model.

Mechlorethamine (HN2) is an alkylating agent and sulfur mustard gas mimetic which is also used in anticancer therapy. HN2 is associated with skin inflammation and blistering which can lead to secondary infections. The purpose of the present study was to investigate the time-dependent dermatotoxicity of HN2 using the mouse ear vesicant model (MEVM). To this end, our operational definition of dermatotoxicity included tissue responses to HN2 consistent with an increase in the wet weights of mouse ear punch biopsies, an increase in the morphometric thickness of H&E stained ear sections and histopathologic observations including tissue edema, inflammatory cell infiltration and vesication. The ears of male Swiss Webster mice were topically exposed to a single dose of HN2 (0.5 µmol/ear) or DMSO vehicle (5 µl/ear) or left untreated (naive). Mice were then euthanized at 15 min, 1, 2, 4, 8 or 24 hr following HN2 exposure. Compared to control ears, mouse ears exposed to HN2 at all time points showed an increase in wet weights, morphometric thickness, edema, inflammatory cell infiltration and signs of vesication. The incidence in tissue vesication sharply increased between 4 and 8 hr after exposure, revealing that tissue vesication is well established by 8 hr and remains elevated at 24 hr after exposure. It is noteworthy that, compared to control ears, mouse ears treated with DMSO vehicle alone also exhibited an increase in wet weights and morphometric thickness at 15 min, 1, 2 and 4 hr following treatment; however, these vehicle effects begin to subside after 4 hr. The results obtained here using the MEVM provide a more holistic understanding of the kinetics of vesication, and indicate that time points earlier than 24 hr may prove useful not only for investigating the complex mechanisms involved in vesication but also for assessing the effects of vesicant countermeasures.

In vitro assessment of the growth and plasma membrane H+ -ATPase inhibitory activity of ebselen and structurally related selenium- and sulfur-containing compounds in Candida albicans.

Ebselen (EB, compound 1) is an investigational organoselenium compound that reduces fungal growth, in part, through inhibition of the fungal plasma membrane H+ -ATPase (Pma1p). In the present study, the growth inhibitory activity of EB and of five structural analogs was assessed in a fluconazole (FLU)-resistant strain of Candida albicans (S2). While none of the compounds were more effective than EB at inhibiting fungal growth (IC50  âˆ¼ 18 µM), two compounds, compounds 5 and 6, were similar in potency. Medium acidification assays performed with S2 yeast cells revealed that compounds 4 and 6, but not compounds 2, 3, or 5, exerted an inhibitory activity comparable to EB (IC50  âˆ¼ 14 µM). Using a partially purified Pma1p preparation obtained from S2 yeast cells, EB and all the analogs demonstrated a similar inhibitory activity. Taken together, these results indicate that EB analogs are worth exploring further for use as growth inhibitors of FLU-resistant fungi.

[Letter to the Editor] Isolation of mitochondria is necessary for precise quantification of mitochondrial DNA damage in human carcinoma samples.

Address correspondence to Carlo Vascotto, Department of Medical and Biological Sciences, University of Udine, Udine, 33100, Italy. E-mail: carlo.vascotto@uniud.it.

A comparative assessment of the cytotoxicity and nitric oxide reducing ability of resveratrol, pterostilbene and piceatannol in transformed and normal mouse macrophages.

The present study investigated the pharmacological effects of three stilbenoids, resveratrol (RES), pterostilbene (PTR) and piceatannol (PIC), in transformed and normal macrophages. Our first aim was to comparatively assess the cytotoxicity of RES, PTR and PIC in unstimulated transformed mouse macrophages (RAW 264.7 cells) and primary peritoneal macrophages (PMs) harvested from both wild type and Nrf2 (nuclear factor erythroid 2-related factor 2)-deficient female mice. Our second aim was to investigate whether the inhibitory effect of RES, PTR and PIC on nitric oxide (NO) release from stimulated PMs depends on the status of the transcription factor Nrf2. The rationale for investigating Nrf2 status was based upon recent reports showing that certain compounds (sulforaphane and linalool) suppress LPS-induced inflammation in an Nrf2-dependent manner. Cell viability studies confirmed our prior work in unstimulated RAW 264.7 cells, with cytotoxic potency decreasing in the order of PTR > PIC > RES. Unstimulated PMs, regardless of Nrf2 status, were less sensitive to stilbenes, requiring at least a threefold higher stilbene concentration to inhibit cell viability, with cytotoxic potency again decreasing in the order of PTR > PIC > RES. In studies focused on our second aim, IC50 values for NO inhibition (measured as [Formula: see text]) in wild type PMs were similar for all three stilbenes (∼10 µM). In Nrf2-deficient PMs, the IC50 for NO inhibition by PIC did not change; however, a rightward shift in the concentration effect curve was observed for both RES and PTR, indicating a role for Nrf2 in the suppression of LPS-induced [Formula: see text] accumulation by these particular stilbenes.

Lipopolysaccharide Attenuates the Cytotoxicity of Resveratrol in Transformed Mouse Macrophages.

Resveratrol and pterostilbene are natural products that are present in plants and have been incorporated into various dietary supplements. Numerous beneficial pharmacologic effects have been reported for these stilbenes; however, the mechanism by which these compounds exert a cytotoxic effect in RAW 264.7 macrophages has not been well characterized. We have previously described that resveratrol is toxic to these tumor-derived macrophages and that stimulation with lipopolysaccharide (LPS) reduces resveratrol toxicity via a mechanism that involves activation of toll like receptor 4. In the present work, we examined the cellular and molecular effects of resveratrol and the related compound pterostilbene by determining cell viability and caspase 3 activity in control and LPS-stimulated RAW 264.7 macrophages incubated with these stilbenes for 24 h. We found that LPS stimulation reduced the cytotoxicity of resveratrol but not of pterostilbene in these cells. When examined for effects on caspase 3 activation after a 24 h incubation, resveratrol and pterostilbene were each found to separately and significantly increase caspase 3 activity in these cells. LPS stimulation prevented caspase 3 activation by pterostilbene and reduced caspase 3 activation by resveratrol in RAW 264.7 macrophages. The data presented here indicate that LPS induces a phenotype switch in tumor-derived RAW 264.7 macrophages in which cells experiencing LPS in the presence of resveratrol or pterostilbene become less likely to activate the pro-apoptotic factor caspase 3.

Ebselen, a Small-Molecule Capsid Inhibitor of HIV-1 Replication.

The human immunodeficiency virus type 1 (HIV-1) capsid plays crucial roles in HIV-1 replication and thus represents an excellent drug target. We developed a high-throughput screening method based on a time-resolved fluorescence resonance energy transfer (HTS-TR-FRET) assay, using the C-terminal domain (CTD) of HIV-1 capsid to identify inhibitors of capsid dimerization. This assay was used to screen a library of pharmacologically active compounds, composed of 1,280in vivo-active drugs, and identified ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], an organoselenium compound, as an inhibitor of HIV-1 capsid CTD dimerization. Nuclear magnetic resonance (NMR) spectroscopic analysis confirmed the direct interaction of ebselen with the HIV-1 capsid CTD and dimer dissociation when ebselen is in 2-fold molar excess. Electrospray ionization mass spectrometry revealed that ebselen covalently binds the HIV-1 capsid CTD, likely via a selenylsulfide linkage with Cys198 and Cys218. This compound presents anti-HIV activity in single and multiple rounds of infection in permissive cell lines as well as in primary peripheral blood mononuclear cells. Ebselen inhibits early viral postentry events of the HIV-1 life cycle by impairing the incoming capsid uncoating process. This compound also blocks infection of other retroviruses, such as Moloney murine leukemia virus and simian immunodeficiency virus, but displays no inhibitory activity against hepatitis C and influenza viruses. This study reports the use of TR-FRET screening to successfully identify a novel capsid inhibitor, ebselen, validating HIV-1 capsid as a promising target for drug development.

Oral delivery of glucagon like peptide-1 by a recombinant Lactococcus lactis.

PURPOSE: To develop a live oral delivery system of Glucagon like peptide-1 (GLP-1), for the treatment of Type-2 Diabetes. METHODS: LL-pUBGLP-1, a recombinant Lactococcus lactis (L. lactis)) transformed with a plasmid vector encoding GLP-1 cDNA was constructed and was used as a delivery system. Secretion of rGLP-1 from LL-pUBGLP-1 was characterized by ELISA. The bioactivity of the rGLP-1 was examined for its insulinotropic activity on HIT-T15 cells. Transport of rGLP-1 across MDCK cell monolayer when delivered by LL-pUBGLP-1 was studied. The therapeutic effect of LL-pUBGLP-1 after oral administration was investigated in ZDF rats. RESULTS: DNA sequencing and ELISA confirmed the successful construction of the LL-pUBGLP-1 and secretion of the active form of rGLP-1. In vitro insulinotropic studies demonstrated that LL-pUBGLP-1 could significantly (p < 0.05) stimulate HIT-T15 cells to secrete insulin as compared to the controls. When delivered by LL-pUBGLP-1, the GLP-1 transport rate across the MDCK cell monolayer was increased by eight times (p < 0.01) as compared to the free solution form. Oral administration of LL-pUBGLP-1 in ZDF rats resulted in a significant decrease (10-20%, p < 0.05) in blood glucose levels during 2-11 h post dosing and a significant increase in insulin AUC0-11h (2.5 times, p < 0.01) as compared to the free solution. CONCLUSION: The present study demonstrates that L. lactis when genetically modified with a recombinant plasmid can be used for the oral delivery of GLP-1.

Selected ebselen analogs reduce mechlorethamine toxicity in vitro.

Sulfur mustard (SM) is a potent vesicant. The lack of an effective antidote makes SM a continued threat to both military and civilian settings. A surrogate agent, namely mechlorethamine (HN2), was used here to mimic the toxicity of SM, and the main objective of this study was to demonstrate if selected organoselenium analogs could protect cultured A-431 skin cells from HN2 toxicity. Test compounds included ebselen (EB-1) and three related organoselenium analogs (EB-2, EB-3 and EB-4). In the absence of test compound, a reproducible and robust cell death was observed in the cells following incubation with HN2 (25 µM, 24 or 48 h) while cells treated with test compound alone (15, 30 or 60 µM) for similar periods of time were generally not affected. When incubated in the presence of both HN2 and test compound for 24 or 48 h, it was found that EB-1, EB-2, EB-3 and EB-4 could spare the cells from death, with the EB-4 compound being the most effective at reducing HN2 toxicity. Light microscopy confirmed these findings. The organoseleniums were also examined for their effects on reducing lipid peroxidation in the A-431 skin cells. Among the test compounds, EB-4 reduced lipid peroxidation by HN2 to the greatest extent. These studies, taken together, validate that the organoselenium antioxidants tested here may serve a purpose in the discovery of medical countermeasures to vesicants.

Use of the mouse ear vesicant model to evaluate the effectiveness of ebselen as a countermeasure to the nitrogen mustard mechlorethamine.

Previous studies in this and other laboratories have demonstrated that ebselen (EB-1), an organoselenium compound, spares cells from mechlorethamine (HN2) toxicity in vitro. In the present study, the hypothesis that EB-1 will reduce dermal toxicity of HN2 in vivo is put forward and found to have merit. Using the mouse ear vesicant model (MEVM), HN2, applied topically, showed a dose-dependent effect upon ear swelling and thickness 24 h after treatment; whereas tissue injury consistent with vesication was observed at the higher test doses of HN2 (≥ 0.250 µmol per ear). To examine HN2 countermeasure activity using the MEVM, either hydrocortisone (HC), as a positive control, or EB-1, the test countermeasure, was administered as three topical treatments 15 min, 4 and 8 h after HN2 exposure. Using this approach, both HC and EB-1 were found to reduce tissue swelling associated with HN2 toxicity 24 h after exposure to the vesicant. Taken together, these data demonstrate for the first time the effectiveness of EB-1 as a vesicant countermeasure in a relevant in vivo model.