Ligand-Substitution Reactions of the Tellurium Compound AS-101 in Physiological Aqueous and Alcoholic Solutions.

Since its first crystallization, the aqueous structure of the tellurium-containing experimental drug AS-101 has never been studied. We show that, under the aqueous conditions in which it is administered, AS-101 is subjected to an immediate ligand-substitution reaction with water, yielding a stable hydrolyzed oxide anion product that is identified, for the first time, to be TeOCl3-. Studying the structure of AS-101 in propylene glycol (PG), an alcoholic solvent often used for the topical and oral administration of AS-101, revealed the same phenomenon of ligand-substitution reaction between the alcoholic ligands. Upon exposure to water, the PG-substituted product is also hydrolyzed to the same tellurium(IV) oxide form, TeOCl3-.

Multifunctional activity of a small tellurium redox immunomodulator compound, AS101, on dextran sodium sulfate-induced murine colitis.

Inflammatory bowel diseases (IBDs) are a group of idiopathic, chronic immune-mediated diseases characterized by an aberrant immune response, including imbalances of inflammatory cytokine production and activated innate and adaptive immunity. Selective blockade of leukocyte migration into the gut is a promising strategy for the treatment of IBD. This study explored the effect of the immunomodulating tellurium compound ammonium trichloro (dioxoethylene-o,o') tellurate (AS101) on dextran sodium sulfate (DSS)-induced murine colitis. Both oral and intraperitoneal administration of AS101 significantly reduced clinical manifestations of IBD. Colonic inflammatory cytokine levels (IL-17 and IL-1ß) were significantly down-regulated by AS101 treatment, whereas IFN-γ was not affected. Neutrophil and α4ß7(+) macrophage migration into the tissue was inhibited by AS101 treatment. Adhesion of mesenteric lymph node cells to mucosal addressin cell adhesion molecule (MAdCAM-1), the ligand for α4ß7 integrin, was blocked by AS101 treatment both in vitro and in vivo. DSS-induced destruction of colonic epithelial barrier/integrity was prevented by AS101, via up-regulation of colonic glial-derived neurotrophic factor, which was found previously to regulate the intestinal epithelial barrier through activation of the PI3K/AKT pathway. Indeed, the up-regulation of glial-derived neurotrophic factor by AS101 was associated with increased levels of colonic pAKT and BCL-2 and decreased levels of BAX. Furthermore, AS101 treatment reduced colonic permeability to Evans blue and decreased colonic TUNEL(+) cells. Our data revealed multifunctional activities of AS101 in the DSS-induced colitis model via anti-inflammatory and anti-apoptotic properties. We suggest that treatment with the small, nontoxic molecule AS101 may be an effective early therapeutic approach for controlling human IBD.

Resolution of inflammation-related apoptotic processes by the synthetic tellurium compound, AS101 following liver injury.

BACKGROUND/AIMS: Fulminant hepatic failure is a dangerous condition, which occurs when large parts of the liver become damaged beyond repair, and the liver is no longer able to function. This syndrome is induced by inflammatory processes, resulting in acute liver failure. Recently, the organotellurium compound, trichloro(dioxoethylene-O,O(')) tellurate (AS101), has been found by our group to be able to directly inhibit caspases, due to its Te(IV)-thiol chemistry. The aim of this study was to examine the potential of AS101 as an anti-inflammatory and anti-apoptotic compound in vitro and in vivo following liver injury. METHODS: Propionibacterium acnes-primed LPS-induced liver injury was performed in Balb/c mice. ALT/AST, cytokines, caspase-1,-3 and-8 activities, and liver histology were assessed. RESULTS: AS101 inhibited TNFalpha or anti-FAS-induced apoptotic processes in hepatocytes in vitro. A P. acnes+LPS in vivo liver injury model revealed lower serum ALT and AST and reduced necrosis and apoptosis in AS101-treated mice. IL-18 and IL-1beta reduced levels in AS101-treated mice were associated with caspase-1 activity inhibition. Our findings suggest IL-6, IL-17 and pSTAT3 as additional novel players in the pathogenicity of FHF. Inhibition of caspase-3, and-8 activities by AS101 treatment contributed to decreased hepatocyte death, resulting in increased survival. CONCLUSIONS: We suggest that due to its interaction with key-target cysteine residues, AS101 mediates anti-inflammatory and anti-apoptotic effects in this FHF model, which may serve as a potent treatment for mitigation of hepatic damage.

A potential antimicrobial treatment against ESBL-producing Klebsiella pneumoniae using the tellurium compound AS101.

Due to the extensive spread of antibiotic-resistant Klebsiella pneumoniae, the non-toxic immunomodulator, ammonium trichloro (dioxoethylene-o, o') tellurate (AS101), was introduced for the first time in this study. Eleven strains of K. pneumoniae were tested: five were extended spectrum beta lactamase (ESBL)-producing strains and six were non-ESBL-producing strains. The MIC and MBC of ten strains were 9 microg/ml AS101 and 18 microg/ml for one strain. AS101 treatment inhibited bacterial growth in a dose-dependent manner on protein-rich media. No inhibition by AS101 was observed on poorer media. In combination with beta-mercaptoethanol (2-ME) or cysteamine, AS101 inhibited bacterial growth in both types of media. Growth inhibition was also shown following AS101 treatment at both lag and log phases. Our data indicate that AS101 enters the bacterium through its porins, causing bacterial destruction. The mechanism of cell death was characterized using several techniques: (a) scanning electron microscopy showed that bacteria treated with AS101 or in combination with cysteamine exhibited evidence of cell-wall damage; (b) X-ray microanalysis demonstrated damage to Na/K pumps; and (c) transmission electron microscopy demonstrated cell lysis. These phenomena suggest that AS101 has antibacterial potential against K. pneumoniae infections.

The cyclin kinase inhibitor p57kip2 regulates TGF-beta-induced compensatory tubular hypertrophy: effect of the immunomodulator AS101.

BACKGROUND: Compensatory tubular cell hypertrophy following unilateral nephrectomy is a cell cycle-dependent process. Our previous study showed that treatment of unilaterally nephrectomized rats with the immunomodulator AS101 partially inhibits compensatory hypertrophy of the remaining kidneys through the inhibition of IL-10-induced TGF-beta secretion by mesangial cells. The present study is focused on understanding the intracellular mechanism(s) of this phenomenon. METHODS: A total of 120 male Sprague-Dawley rats were unilaterally nephrectomized or sham-operated and treated with AS101 or PBS. Kidney weight and protein/DNA ratio were assessed for each experimental animal. The expression of TGF-beta, PCNA, CDK 2, pRb, ppRb, p21(Waf1), p27(kip1) and p57(kip2) proteins in renal tissues was determined by western blot analysis and immunohistochemistry, and the immunoprecipitation of cyclin E complexes was performed. RESULTS: Compensatory renal growth is initiated by proliferation of resident renal cells that precedes hypertrophy. Changes in TGF-beta expression were positively correlated with the amounts of p57(kip2), but not with p21(Waf1) and p27(kip1) expression in the remaining kidneys. Moreover, there was a marked abundance of p57(kip2) but not p21(Waf1) and p27(kip1) binding to the cyclin E complex in PBS-treated unilaterally nephrectomized rats compared to sham-operated animals. Treatment of uninephrectomized rats with AS101 reduced kidney weight and protein/DNA ratio, inhibited TGF-beta and p57(kip2) expression in the remaining kidneys, and decreased the level of p57(kip2) binding to cyclin E complexes. CONCLUSION: These results demonstrate that TGF-beta-induced compensatory tubular cell hypertrophy is regulated in vivo by p57(kip2) but not by the p21(Waf1) and p27(kip1) cyclin kinase inhibitor proteins.

Multifunctional tellurium molecule protects and restores dopaminergic neurons in Parkinson's disease models.

In Parkinson's disease (PD) dopaminergic neurons in the substantia nigra (SN) become dysfunctional and many ultimately die. We report that the tellurium immunomodulating compound ammonium trichloro(dioxoethylene-O,O'-)tellurate (AS101) protects dopaminergic neurons and improves motor function in animal models of PD. It is effective when administered systemically or by direct infusion into the brain. Multifunctional activities of AS101 were identified in this study. These were mainly due to the peculiar Tellur(IV)-thiol chemistry of the compound, which enabled the compound to interact with cysteine residues on both inflammatory and apoptotic caspases, resulting in their inactivation. Conversely, its interaction with a key cysteine residue on p21(ras), led to its activation, an obligatory activity for AS101-induced neuronal differentiation. Furthermore, AS101 inhibited IL-10, resulting in up-regulation of GDNF in the SN. This was associated with activation of the neuroprotective kinases Akt and mitogen-activated protein kinases, and up-regulation of the antiapoptotic protein Bcl-2. Inhibition of caspase-1 and caspase-3 activities were associated with decreased neuronal death and inhibition of IL-1beta. We suggest that, because multiple mechanisms are involved in the dysfunction and death of neurons in PD, use of a multifunctional compound, exerting antiapoptotic, anti-inflammatory, and neurotrophic-inducing capabilities may be potentially efficacious for the treatment of PD.

The synthetic tellurium compound, AS101, is a novel inhibitor of IL-1beta converting enzyme.

The organotellurium compound, trichloro(dioxoethylene-O,O') tellurate (AS101) has been shown previously to exert diverse biologic activities both in vitro and in vivo. This compound was recently found to react with thiols and to catalyze their oxidation. This property of AS101 raises the possibility that it may serve as a cysteine protease inhibitor. In the present study, using a substrate-specific enzymatic assay, we show that treatment of caspase-1 (interleukin-1beta [IL-1beta] converting enzyme [ICE]) with AS101 inhibits its enzymatic activity in a dose-dependent manner. Moreover, the results show that AS101 treatment causes a significant reduction in the active form of IL-18 and IL-1beta in peripheral blood mononuclear cells (PBMC) and in human HaCat keratinocytes. We further demonstrate that the inhibitory effect of AS101 does not involve nitric oxide (NO) or interferon-gamma (IFN-gamma), two possible regulators of IL-18 production, and does not occur at the mRNA level, suggesting a posttranscriptional mechanism of action. More importantly, AS101 downregulates IL-18 and IL-1beta serum levels in a mouse model of lipopolysaccharide (LPS)-induced sepsis, resulting in increased survival. Recent studies emphasize the pathophysiologic role of IL-18 and IL-1beta in a variety of inflammatory diseases. Thus, their blockage by the nontoxic compound, AS101, currently used in clinical studies, may provide clinical advantage in the treatment of these diseases.

The organotellurium compound ammonium trichloro(dioxoethylene-o,o')tellurate reacts with homocysteine to form homocystine and decreases homocysteine levels in hyperhomocysteinemic mice.

Ammonium trichloro(dioxoethylene-o,o')tellurate (AS101) is an organotellurium compound with pleiotropic functions that has been associated with antitumoral, immunomodulatory and antineurodegenerative activities. Tellurium compounds with a +4 oxidation state, such as AS101, react uniquely with thiols, forming disulfide molecules. In light of this, we tested whether AS101 can react with the amino acid homocysteine both in vitro and in vivo. AS101 conferred protection against homocysteine-induced apoptosis of HL-60 cells. The protective mechanism of AS101 against homocysteine toxicity was directly mediated by its chemical reactivity, whereby AS101 reacted with homocysteine to form homocystine, the less toxic disulfide form of homocysteine. Moreover, AS101 was shown here to reduce the levels of total homocysteine in an in vivo model of hyperhomocysteinemia. As a result, AS101 also prevented sperm cells from undergoing homocysteine-induced DNA fragmentation. Taken together, our results suggest that the organotellurium compound AS101 may be of clinical value in reducing total circulatory homocysteine levels.

Novel involvement of the immunomodulator AS101 in IL-10 signaling, via the tyrosine kinase Fer.

Interleukin-10 (IL-10) plays a major proliferative role in many tumors, and activates the transcription factor Stat3 by tyrosine phosphorylation. The immunomodulator ammonium trichloro (dioxoethylene-o,o') tellurate (AS101) has a direct antitumor activity, and is able to sensitize several tumors to chemotherapy, by inhibiting the tumor IL-10 autocrine loop. The tyrosine kinase Fer is essential for the proliferation of numerous malignant cell lines and in some cases was related to Stat3 activation. This article examined the role of AS101 in IL-10 signaling, and the correlation between Fer and Stat3, in human peripheral blood mononuclear cells (PBMC). We show that Fer was associated with Stat3 in PBMC and RAW 264.7, a macrophage cell line. Recombinant IL-10 (rIL-10) increased the tyrosine phosphorylation of Stat3, upregulated the levels of Fer, and increased the association of Fer with phosphorylated Stat3 (pYStat3). All the activities of IL-10 mentioned above were reversed by AS101. The effects conferred by AS101 were totally abolished by exogenous addition of rIL-10. These results indicate that AS101 downregulates the Stat3 IL-10 loop, and inhibits Fer association with pYStat3. We conclude that anti-IL-10 treatment using AS101, may be beneficial in certain malignancies and other pathologies in which IL-10 secretion is elevated and Stat3 is continuously phosphorylated.

Upregulation of carp GDNF mRNA by the immunomodulator AS101.

Although the glia derived neurotrophic factor (GDNF) is defined as a molecule that maintains neuronal cells, it possesses a range of functions outside the nervous system. For example, it is essential for uretric branching in kidney morphogenesis and for regulating the differentiation of stem cells during spermatogenesis, cardiac, hair follicle and vascular differentiation and the maintenance of immune cells. In the present work, the presence of GDNF in carp peripheral blood leukocytes (PBL) and head kidney cells (HK) was evidenced and its evolutionary importance in both neural and immune systems development was suggested. Using the northern-blot technique, we could observe the expression of two different transcripts of this gene. GDNF upregulation was detected using semi-quantitative PCR, following ex vivo treatment of PBL and HK cells with the immunomodulator AS101 which was previously shown to inhibit IL-10 and to up-regulate GDNF protein levels in human SVG astrocyte cell line, in 6-OHDA hemi-parkinsonian mice in vivo and in rat glomerular mesengial cells in vitro.

The immunomodulator AS101 induces growth arrest and apoptosis in multiple myeloma: association with the Akt/survivin pathway.

Multiple Myeloma (MM) is a clonal B-cell malignancy affecting both the immune and the skeletal systems, and accounts for 10% of all hematological cancers. The immunomodulator ammonium trichloro (dioxoethylene-O,O') tellurate (AS101) is a non-toxic compound which has direct anti-tumoral properties in several tumor models. The present study examined the anti-tumoral activity of AS101 in MM by targeting the Akt/Survivin signaling pathway, crucial for survival. We showed that AS101 inhibites cell proliferation and colonies formation of MM cell lines, in a dose-dependent manner. AS101 induced G(2)/M growth arrest and increased both cyclin-dependent kinase inhibitor p21(waf1) protein levels and Cdk1 (p34(cdc2))-inhibitory phosphorylation. Longer incubation of MM cells with AS101 resulted in accumulation of apoptotic cell population and in increased caspase 9, 3 and 7 activities. We also showed that AS101 down-regulated Akt phosphorylation and decreased expression of the inhibitor of apoptosis, survivin. Since Akt and survivin are potentials targets for MM therapy, we suggest that AS101, currently being used in clinical studies, may have therapeutic implications in myeloma and other hematopoietic malignancies.

Ammonium trichloro(dioxoethylene-o,o')tellurate (AS101) sensitizes tumors to chemotherapy by inhibiting the tumor interleukin 10 autocrine loop.

Cancer cells of different solid and hematopoietic tumors express growth factors in respective stages of tumor progression, which by autocrine and paracrine effects enable them to grow autonomously. Here we show that the murine B16 melanoma cell line and two human primary cultures of stomach adenocarcinoma and glioblastoma multiforme (GBM) constitutively secrete interleukin (IL)-10 in an autocrine/paracrine manner. This cytokine is essential for tumor cell proliferation because its neutralization decreases clonogenicity of malignant cells, whereas addition of recombinant IL-10 increases cell proliferation. The immunomodulator ammonium trichloro(dioxoethylene-o,o')tellurate (AS101) decreased cell proliferation by inhibiting IL-10. This activity was abrogated by exogenous addition of recombinant IL-10. IL-10 inhibition by AS101 results in dephosphorylation of Stat3, followed by reduced expression of Bcl-2. Moreover, these activities of AS101 are associated with sensitization of tumor cells to chemotherapeutic drugs, resulting in their increased apoptosis. More importantly, AS101 sensitizes the human aggressive GBM tumor to paclitaxel both in vitro and in vivo by virtue of IL-10 inhibition. AS101 sensitizes GBM cells to paclitaxel at concentrations that do not affect tumor cells. This sensitization can also be obtained by transfection of GBM cells with IL-10 antisense oligonucleotides. Sensitization of GBM tumors to paclitaxel (Taxol) in vivo was obtained by either AS101 or by implantation of antisense IL-10-transfected cells. The results indicate that the IL-10 autocrine/paracrine loop plays an important role in the resistance of certain tumors to chemotherapeutic drugs. Therefore, anti-IL-10 treatment modalities with compounds such as AS101, combined with chemotherapy, may be effective in the treatment of certain malignancies.

Hair growth induction by the Tellurium immunomodulator AS101: association with delayed terminal differentiation of follicular keratinocytes and ras-dependent up-regulation of KGF expression.

The synthetic immunomodulator AS101[ammonium trichloro(dioxoethylene-o,o')tellurate] was previously found to protect cancer patients from chemotherapy-induced bone marrow toxicity and alopecia. Here we show that AS101 induces hair growth in nude and normal mice. AS101 possesses the dual ability to both induce anagen and retard spontaneous catagen in the C57BL/6 mouse model. Anagen induced by AS101 is mediated by keratinocyte growth factor (KGF), as it is abrogated both in nude mice co-treated with AS101 plus neutralizing anti KGF antibodies and in AS101-treated transgenic mice expressing a dominant-negative KGF receptor transgene in basal keratinocytes. AS101 up-regulates KGF expression by activating the ras signaling pathway in cultured fibroblasts. AS101-induced delayed catagen is associated with inhibition of terminal differentiation marker expression both in nude and C57BL/6 mice epidermal follicular keratinocytes and in cultures of primary mouse follicular keratinocytes induced to differentiate. This activity is associated with relatively sustained elevation of p21waf. Delayed expression of terminal differentiation markers was not induced by AS101 in follicular keratinocytes from p21waf knockout mice. Because similar results were obtained with cultures of primary human keratinocytes and fibroblasts, preliminary case report studies revealed substantial hair growth when AS101 was topically applied on three adolescents who had remained alopeciac 1-2 years after chemotherapy. The results emphasize the unique mode of action of AS101 and highlight its potential clinical use for treating certain types of alopecia.

Sensitizing B- and T- cell Lymphoma Cells to Paclitaxel/Abraxane-Induced Death by AS101 via Inhibition of the VLA-4-IL10-Survivin Axis.

UNLABELLED: Cancer cell resistance to chemotherapy is a major concern in clinical oncology, resulting in increased tumor growth and decreased patient survival. Manipulation of apoptosis has emerged as a new therapeutic strategy to eliminate cancer cells. The focus of this study resides within a novel approach to target survivin, an integrator of both cell death and mitosis. This protein plays a pivotal role in the resistance of tumors to chemotherapy, especially to paclitaxel. The data herein demonstrate an indirect repression of survivin in both B- and T-cell lymphoma and human NHL by the nontoxic tellurium compound, AS101 [ammonium trichloro(dioxoethylene-o,o')tellurate], via inhibition of tumor autocrine IL10-STAT3-Survivin signaling. As a result of survivin abrogation, sensitization of lymphomas to paclitaxel or to Abraxane, the new albumin-stabilized nanoparticle formulation of paclitaxel, occurs both in vitro and in vivo. Importantly, inhibition of lymphoma cell IL10 secretion is mediated by inactivation of the VLA-4 integrin, recently shown to be an important target of AS101. This activity is followed by inhibition of the PI3K-AKT axis that mediates IL10 suppression. Because a wide variety of lymphomas and other tumor types express VLA-4 and secrete IL10 in an autocrine manner, inhibition of survivin with a small nontoxic agent has vast clinical significance in modulating chemosensitivity in many tumor types. IMPLICATIONS: Combination therapy with AS101 and paclitaxel has novel therapeutic potential targeting deregulated active pathways in lymphoma, overcoming endogenous resistance to apoptosis.

Redox modulation of adjacent thiols in VLA-4 by AS101 converts myeloid leukemia cells from a drug-resistant to drug-sensitive state.

Interaction between the integrin VLA-4 on acute myelogenous leukemia (AML) cells with stromal fibronectin is a decisive factor in chemotherapeutic resistance. In this study, we provide a rationale for a drug repositioning strategy to blunt integrin activation in AML cells and restore their sensitivity to chemotherapy. Specifically, we demonstrate that the nontoxic tellurium compound AS101, currently being evaluated in clinical trials, can abrogate the acquired resistance of AML. Mechanistic investigations revealed that AS101 caused redox inactivation of adjacent thiols in the exofacial domain of VLA-4 after its ligation to stromal fibronectin. This effect triggered cytoskeletal conformational changes that decreased PI3K/Akt/Bcl2 signaling, an obligatory step in chemosensitization by AS101. In a mouse xenograft of AML derived from patient leukemic cells with high VLA-4 expression and activity, we demonstrated that AS101 abrogated drug resistance and prolonged survival in mice receiving chemotherapy. Decreased integrin activity was confirmed on AML cells in vivo. The chemosensitizing activity of AS101 persisted in hosts with defective adaptive and innate immunity, consistent with evidence that integrin deactivation was not mediated by heightening immune attack. Our findings provide a mechanistic rationale to reposition the experimental clinical agent, AS101, to degrade VLA-4-mediated chemoresistance and improve clinical responses in patients with AML.

The impact of light source on discrimination ability in subjects with age-related macular degeneration.

PURPOSE: To examine the influence of light source on letter contrast sensitivity in subjects with age-related macular degeneration (AMD). METHODS: Halogen incandescent bulbs and low-energy fluorescent tubes were tested with 70 subjects with AMD. The subjects' contrast sensitivity was determined in a randomized single-blind crossover study for each light source using photopically illuminated Pelli Robson contrast sensitivity charts. The test subjects' subjective light source preference was also determined. RESULTS: The mean contrast sensitivity for the incandescent light source was 1.28 ± 0.29 (mean ± SD), and for the fluorescent light source 1.17 ± 0.29, p < 0.001. The illuminance was 338 lux (± 9) for the incandescent light, and 339 lux (± 11) for the fluorescent light. Forty-nine subjects preferred the incandescent light source, while none preferred the fluorescent light source for maximum detail and clarity. Nineteen had no preference. This finding is statistically significant. Fifteen of the 19 subjects without a preference had no difference in contrast sensitivity, which supports their lack of preference. There was no significant difference with regard to sex or order of exposure to light source. Subjects with AMD had significantly reduced contrast sensitivity compared with expected normal values. We found no relationship between visual acuity and contrast sensitivity. CONCLUSION: We are only able to recommend photopic full spectral radiance incandescent light sources to visually impaired subjects for their domestic surroundings. Furthermore, we recommend the use of full spectral radiance light sources for the illumination of Pelli-Robson contrast sensitivity charts. Given equal illuminance, as in our study, the findings show that contrast sensitivity was better by illumination with incandescent light with full spectral radiance compared with fluorescent light with interrupted spectral radiance.

The anti-inflammatory effects of the tellurium redox modulating compound, AS101, are associated with regulation of NFkappaB signaling pathway and nitric oxide induction in macrophages.

BACKGROUND: LPS-activated macrophages produce mediators which are involved in inflammation and tissue injury, and especially those associated with endotoxic shock. The non toxic tellurium compound ammonium tri-chloro(dioxoethylene-O,O'-)tellurate, AS101, has been recently shown to exert profound anti-inflammatory properties in animal models, associated with its Te(IV) redox chemistry. This study explores the anti-inflammatory properties of AS101 with respect to modulation of inflammatory cytokines production and regulation of iNOS transcription and expression in activated macrophages via targeting the NFkB complex. RESULTS: AS101 decreased production of IL-6 and in parallel down-regulated LPS-induced iNOS expression and NO secretion by macrophages. AS101 reduced IkB phosphorylation and degradation, and reduced NFkB nuclear translocalization, albeit these effects were exerted at different kinetics. Chromatin immunoprecipitation assays showed that AS101 treatment attenuated p50-subunit ability to bind DNA at the NFkB consensus site in the iNOS promotor following LPS induction. CONCLUSIONS: Besides AS101, the investigation of therapeutic activities of other tellurium(IV) compounds is scarce in the literature, although tellurium is the fourth most abundant trace element in the human body. Since IKK and NFkB may be regulated by thiol modifications, we may thus envisage, inview of our integrated results, that Te(IV) compounds, may have important roles in thiol redox biological activity in the human body and represent a new class of anti-inflammatory compounds.