Michael Albeck

Anti-IL-10 Therapeutic Strategy Using the Immunomodulator AS101 in Protecting Mice from Sepsis-Induced Death: Dependence on Timing of Immunomodulating Intervention

The role of IL-10 in experimental sepsis is controversial. The nontoxic immunomodulator, ammonium trichloro(dioxoethylene-o,o′)tellurate (AS101) has been previously shown to inhibit IL-10 expression at the transcriptional level. In this study, we show that in mice subjected to cecal ligation and puncture (CLP), treatment with AS101 12 h after, but not before, CLP significantly increased survival of septic mice. This was associated with a significant decrease in serum IL-10 and in IL-10 secretion by peritoneal macrophages 24–48 h after CLP. At that time, the ability of these cells to secrete TNF-α and IL-1β was restored in AS101-treated mice. The increased survival of AS101-treated mice was due to the inhibition of IL-10, since cotreatment with murine rIL-10 abolished the protective activity of AS101. AS101 increased class II Ag expression on peritoneal macrophages, severely depressed in control mice, while it did not affect the expression of class I Ags. This was accompanied by a significant elevation in the level of IFN-γ secreted by splenocytes. Moreover, AS101 ameliorated bacterial clearance in the peritoneum and blood and decreased severe multiple organ damage, as indicated by clinical chemistry. Furthermore, myeloperoxidase levels in the liver and lung of AS101-treated mice, an indirect means of determining the recruitment of neutrophils, were significantly decreased. We suggest that nontoxic agents such as AS101, with the capacity to inhibit IL-10 and stimulate macrophage functions, may have clinical potential in the treatment of sepsis, provided they are administered during the phase of sepsis characterized by immune suppression.

The protective role of the immunomodulator AS101 against chemotherapy‐induced alopecia studies on human and animal models

The immunomodulator AS101 has been demonstrated to exhibit radioprotective and chemoprotective effects in mice. Following phase‐I studies, preliminary results from phase‐II clinical trials on non‐small‐cell‐lung‐cancer patients showed a reduction in the severity of alopecia in patients treated with AS101 in combination with chemotherapy. To further substantiate these findings, the present study was extended to include 58 patients treated either with the optimal dose of 3 mg/m2 AS101 combined with carboplatin and VP‐16, or with chemotherapy alone. As compared with patients treated with chemotherapy alone, there was a significant decrease in the level of alopecia in patients receiving the combined therapy. The newly developed rat model was used to elucidate the protective mechanism involved in this effect. We show that significant prevention of chemotherapy‐induced alopecia is obtained in rats treated with Ara‐C combined with AS101, administered i.p. or s.c. or applied topically to the dorsal skin. We show that this protection by AS101 is mediated by macrophage‐derived factors induced by AS101. Protection by AS101 can be ascribed, at least in part, to IL‐1, since treatment of rats with IL‐1RA largely abrogated the protective effect of AS101. Moreover, we demonstrate that in humans there is an inverse correlation between the grade of alopecia and the increase in IL‐1α. In addition, protection by AS101 could be related to PGE2 secretion, since injection of indomethacin before treatment with AS101 and Ara‐C partly abrogated the protective effect of AS101. To assess the ability of AS101 to protect against chemotherapy‐induced alopecia, phase‐II clinical trials have been initiated with cancer patients suffering from various malignancies.

Bone marrow-sparing and prevention of alopecia by AS101 in non-small-cell lung cancer patients treated with carboplatin and etoposide.

PURPOSE The aim of this study was to evaluate the ability of the immunomodulator AS101 to prevent chemotherapy-induced neutropenia and thrombocytopenia and thus allow patients to receive full-dose antineoplastic agents according to protocol design. We also aimed to determine the production level of various hematopoietic growth factors in treated patients. PATIENTS AND METHODS This study of 44 unresectable or metastatic non-small-cell lung cancer (NSCLC) patients was an open-label prospective randomized study of standard chemotherapy alone versus chemotherapy plus AS101. Each patient received carboplatin (300 mg/m2 intravenously [IV] on day 1 of a 28-day cycle, and etoposide (VP-16) (200 mg/m2 orally) on days 3, 5, and 7 of each cycle. AS101 was administered at 3 mg/m2 three times per week starting 2 weeks before chemotherapy. RESULTS AS101, which manifested no major toxicity, significantly reduced neutropenia and thrombocytopenia and thus allowed all treated patients to receive full-dose antineoplastic agents, in contrast to only 28.5% of the control group. Continuous treatment with AS101 significantly reduced the number of days per patient of thrombocytopenia and neutropenia and did not provide protection to tumor cells as reflected by the higher overall response rate compared with the chemotherapy-alone arm. Interestingly, AS101 treatment also significantly prevented chemotherapy-induced alopecia. These effects correlate with the ability of AS101-treated patients to increase significantly the production of colony-stimulating factors (CSFs) interleukin-1 alpha (IL-1 alpha) and IL-6. CONCLUSION AS101 has significant bone marrow (BM)-sparing effects and prevents hair loss in chemotherapy-treated patients, with minimal overall toxicity. These effects are probably due to increased production of IL-1 alpha, IL-6, and granulocyte-macrophage (GM)-CSF.

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.

The organotellurium compound ammonium trichloro(dioxoethylene‐0,0′) tellurate enhances neuronal survival and improves functional outcome in an ischemic stroke model in mice

Ammonium trichloro(dioxoethylene‐0,0′) tellurate (AS101) is a non‐toxic organotellurium compound with pleiotropic activities. It was recently shown to induce production of the neurotrophic factor glial cell line‐derived neurotrophic factor and to rescue neuronal‐like PC‐12 cells from neurotrophic factor deprivation‐induced apoptosis. In this study, we show that AS101 improves functional outcome and reduces brain damage in a mouse model of focal ischemic stroke. Both pre‐stroke and post‐stroke intraperitoneal treatments with AS101 reduced infarct size and edema and improved the neurological function of the animals. AS101 treatments reduced both apoptotic and inflammatory caspase activities, and also inhibited protein tyrosine nitration suggesting that AS101 suppresses oxidative stress. Studies of cultured neurons showed that AS101 confers protection against apoptosis induced by either glucose deprivation or the lipid peroxidation product 4‐hydroxynonenal. Moreover, AS101 treatment reduced glutamate‐induced intracellular calcium elevation, a major contributor to neuronal death in stroke. As AS101 has an excellent safety profile in humans, our pre‐clinical data suggest a potential therapeutic benefit of AS101 in patients suffering from stroke and other neurodegenerative conditions.

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.

Synergistic anti-tumoral effect of paclitaxel (taxol)+AS101 in a murine model of B16 melanoma: Association with ras-dependent signal-transduction pathways

Optimal doses of paclitaxel (Taxol) combined with the immunomodulator AS101, previously shown to have anti‐tumoral effects, administered to B16 melanoma‐bearing mice decreased tumor volume and resulted in over 60% cure. Paclitaxel+AS101 directly inhibited the clonogenicity of B16 melanoma cells in a synergistic, dose‐dependent manner. We suggest that this results from both reduced paclitaxel‐induced bone marrow toxicity and induction of differential signal‐transduction pathways, which lead to apoptosis of tumor cells. Paclitaxel+AS101 synergistically activated c‐raf‐1 and MAPK ERK1 and ERK2. This activation was essential for the synergistic induction of p21waf protein. Cell‐cycle analysis of B16 cells treated with both compounds revealed an increased accumulation in G2M, though AS101 alone produced significant G1 arrest. These activities were ras‐dependent. AS101+paclitaxel induced significant synergistic phosphorylation (inactivation) of the anti‐apoptotic protein Bcl‐2. Whereas phosphorylation of Bcl‐2 by paclitaxel was raf‐dependent only, the synergistic effect of both compounds together was ras‐, raf‐ and MAPK‐dependent. No effect of the combined treatment on Bax protein expression was observed. We suggest that AS101 renders more cells susceptible to Bcl‐2 phosphorylation by paclitaxel, possibly by increasing the accumulation of paclitaxel‐induced cells in G2M. Exposure of B16 cells to clinically achievable concentrations of paclitaxel+AS101 increased the rate of apoptosis of treated cells. Apoptosis induced by AS101 alone was both raf‐ and MAPK‐dependent, while that induced by paclitaxel was raf‐dependent only. Int. J. Cancer 86:281–288, 2000.

Protective effect of the immunomodulator AS101 against cyclophosphamide-induced testicular damage in mice

BACKGROUND Cyclophosphamide (Cy), a widely used anticancer drug, is associated with significant testicular damage and sterility. Co-administration of the immunomodulating compound AS101 during chemotherapy treatments was previously shown to protect organs against cytotoxic damage, without attenuating the drugs anticancer effect. In this animal study, we investigated the effect of AS101 on testicular damage, sperm DNA damage and infertility induced by Cy. Akt and glycogen synthase kinase-3beta (GSK-3beta) phosphorylation were investigated as a possible chemoprotective mechanism. METHODS Mature male mice, 10 in each group, were injected intraperitoneally with 200 mg/kg Cy once a week for 5 weeks, with or without concurrent treatment with 10 microg per mouse AS101 three times per week. Damage to testicular tubules and sperm production was determined, sperm chromatin damage was analyzed and fertility was gauged. Akt and GSK-3beta phosphorylation were evaluated. RESULTS Co-treatment with AS101 during the course of Cy administration significantly reduced the percentage of damaged seminiferous tubules (76.0 +/- 10.8% versus 40.3 +/- 2.6%), and reduced sperm DNA fragmentation (%DFI) from 44.7 +/- 1.0% to 25 +/- 6.5%. Co-treatment with AS101 also partially protected against the decrease in numbers of impregnated females and litter size. AS101 increased Akt and GSK-3beta phosphorylation. CONCLUSIONS Our results indicate that AS101 can significantly protect against Cy-induced testicular damage and sperm DNA damage, probably by acting through Akt/GSK-3beta phosphorylation.

Toxicity study in rats of a tellurium based immunomodulating drug, AS-101: A potential drug for AIDS and cancer patients

Male and female Sprague Dawley rats were injected intraperitoneally for 4 weeks with ammonium trichloro (dioxyethylene-0-0′-) tellurate, an immunomodulating drug at closes ranging from 3 to 24 mg/kg/week. Routine laboratory examinations included body weight, food consumption, clinical chemistry and hematological examinations. At termination of the experiment, all rats were sacrificed and subjected to a detailed necropsy. Few mortalities were recorded during the course of the study. Clinical signs included hind limb paresis and paraphimosis. A garlic odor pervaded the room. Body weight and food consumption were adversely affected in a dose-related manner. Effects were elicited on the hematological system; changes being noted in the platelet and leukocyte counts as well. Clinical chemistry evaluation revealed signs of hepatoxicity, especially in the female treated groups. The level of beta-globulin was increased. At necropsy organs were found to have a grayish-blue discoloration. Tellurium related histopathological changes were observed in the eyes, liver, thymus, bone marrow, heart and kidneys. An attempt has been made to compare the toxicity of this drug with other tellurium-containing compounds. A good correlation was found. Novel effects of the drug were retinopathy and replacement of bone marrow by bony or fibrous tissue. The possibility that some of the effects may have been elicited due to selenium-vitamin E deficiency has been considered.

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.