Glenn M. Swartz

2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF.

Inhibition of angiogenesis is an important new modality for cancer treatment. 2-methoxyestradiol (2ME2) is a novel antitumor and antiangiogenic agent, currently in clinical trials, whose molecular mechanism of action remains unclear. Herein, we report that 2ME2 inhibits tumor growth and angiogenesis at concentrations that efficiently disrupt tumor microtubules (MTs) in vivo. Mechanistically, we found that 2ME2 downregulates hypoxia-inducible factor-1 (HIF) at the posttranscriptional level and inhibits HIF-1-induced transcriptional activation of VEGF expression. Inhibition of HIF-1 occurs downstream of the 2ME2/tubulin interaction, as disruption of interphase MTs is required for HIF-alpha downregulation. These data establish 2ME2 as a small molecule inhibitor of HIF-1 and provide a mechanistic link between the disruption of the MT cytoskeleton and inhibition of angiogenesis.

2-Methoxyestradiol: an endogenous antiangiogenic and antiproliferative drug candidate.

Abstract2-Methoxyestradiol, once considered an inacitve end-metabolite of estradiol, has recently emerged as a very promising agent for cancer treatment. It is orally active in a wide range of tumor models, and inhibits tumor growth at doses showing non clinical signs of toxicity. 2ME2 targets both the tumor cell and endothelial cell compartments by inducing apoptosis in rapidly proliferating cells and inhibiting blood vessel formation at several stages in the angiogenic cascade. Moreover, the ability of 2ME2 to inhibit metastic spread in several models adds to its therapeutic value for cancer treatment at various stages of the disease. Though the mechanism of action is still undefined, several potential molecular targets and pathways of activation have been suggested.

2-Methoxyestradiol Inhibits Hypoxia-Inducible Factor 1α, Tumor Growth, and Angiogenesis and Augments Paclitaxel Efficacy in Head and Neck Squamous Cell Carcinoma

Purpose: Head and neck squamous cell carcinomas have been reported to overexpress hypoxia-inducible factor (HIF)-1α, a transcription factor that promotes expression of angiogenesis factors and resistance to programmed and therapy-induced cell death. 2-Methoxyestradiol (2ME2) is a natural compound with HIF-1α inhibitory activity that is currently being evaluated in phase 1 and 2 clinical trials for advanced solid tumors and multiple myeloma. To our knowledge, this is the first study to evaluate the effects of 2ME2 in head and neck squamous cell carcinoma. Experimental Design: In the present study, we investigated the effects of 2ME2 alone and in combination with paclitaxel, an active agent in recurrent or advanced head and neck squamous cell carcinoma. Results: 2ME2 exhibited antiproliferative and cytotoxic effects in a panel of five head and neck squamous cell carcinoma cell lines in the 0.5 to 10 μmol/L range, including induction of G2-M blockade, caspase-3/7 activation, and apoptosis at 48 hours. 2ME2 resulted in decreased nuclear HIF-1α–binding activity and affected the expression of downstream genes, such as bid, a proapoptotic bcl-2 family member, and vascular endothelial growth factor, a proangiogenic cytokine. The up-regulation of Bid (57.5% at 12 hours, P < 0.0006) and inhibition of vascular endothelial growth factor secretion (57.7% at 24 hours, P < 0.015; and 50.3% at 48 hours, P < 0.0006) could be partially attributed to the effects on HIF-1α, because HIF-1α small interfering RNAs produced similar effects. Finally, in vivo, in a xenograft model of head and neck squamous cell carcinoma using UM-SCC-11A cells, 2ME2 exhibited antitumor and antiangiogenic activity, as measured by CD31 immunostaining. Conclusions: These results provide support for the use of 2ME2 in combination with paclitaxel for the treatment of recurrent or advanced head and neck squamous cell carcinoma.

Liposomes in leishmaniasis: Therapeutic effects of antimonial drugs, 8-aminoquinolines, and tetracycline

Abstract This study investigated the role of liposomes in changing the pharmacological efficacy of anti-leishmanial drugs in hamsters infected with visceral leishmaniasis. Enhanced anti-leishmanial activity could be accounted for only by liposome-encapsulated drugs. “Empty” liposomes (lacking anti-leishmanial drug) gave no therapeutic benefit by themselves, nor did they enhance the effectiveness of concurrently administered drugs. In the absence of additional drugs, empty liposomes actually resulted in a higher mortality due to endstage leishmaniasis. Mortality associated with chronic leishmaniasis, including that induced by empty liposomes, was reduced approximately 50% by orally administered unencapsulated tetracycline. Liposome-encapsulated tetracycline, given i.c., had no anti-leishmanial activity, thus indicating that tetracycline did not have inherent anti-leishmanial properties, and was beneficial because of its anti-bacterial effects. Liposomes containing an antimonial drug were effective when given i.c., i.p., or i.m., but not when given s.c. or p.o. Liposome-encapsulated antimonial drug had prophylactic activity and was effective when administered 8 days prior, but not 17 days prior, to infection. Unencapsulated antimonial drug had no prophylactic effect. In addition to antimonials, another class of compounds, 8-aminoquinolines, had marked anti-leishmanial activity in liposomes. One of these, WR 6026, was 700 to 1800 times more effective than an antimonial drug alone.

Immunization with cholesterol-rich liposomes induces anti-cholesterol antibodies and reduces diet-induced hypercholesterolemia and plaque formation

Immunization of rabbits with a protein-free formulation consisting of liposomes containing 71% cholesterol and lipid A as an adjuvant induced anticholesterol antibodies that caused complement-dependent lysis of liposomes lacking lipid A. The antibodies, immunoglobulin G (IgG) and immunoglobulin M (IgM), also recognized nonoxidized crystalline cholesterol as an antigen by enzyme-linked immunosorbent assay (ELISA). The effects of immunization against cholesterol on elevations in serum cholesterol and development of atherosclerosis were examined in rabbits fed a diet containing 0.5% to 1.0% cholesterol. Although the mean serum cholesterol level, mainly in the form of very-low-density lipoprotein cholesterol, rose as much as 60-fold in the nonimmunized rabbits, the elevation was significantly less--as much as 35% lower--in the immunized rabbits. Elevation of serum cholesterol was accompanied by an apparent drop in the level of antibodies on initiating the diet, followed by a rebound on stopping the diet, thus suggesting that the antibodies were adsorbed to cholesterol that was present in circulating lipoproteins. When lipoprotein fractions--composed of either very-low-density and intermediate-density lipoproteins derived from cholesterol-fed nonimmunized rabbits or human low-density lipoproteins--were tested as capture antigens by solid-phase ELISA, reactivity was observed with IgG and IgM antibodies present in the serum of immunized rabbits. Immunization also resulted in a marked decrease in the risk of developing atherosclerosis. Analysis of aortic atherosclerosis by quantitative histologic examination and fatty streaks by automated morphometric probability-of-occurrence mapping showed diminished atherosclerosis in most areas of the aorta in vaccine recipients. It is proposed that immunization with liposomes containing 71% cholesterol and lipid A can reduce diet-induced hypercholesterolemia and atherosclerosis.

Significant antitumor activity in vivo following treatment with the microtubule agent ENMD-1198

Clinical studies using the microtubule-targeting agent 2-methoxyestradiol (2ME2; Panzem) in cancer patients show that treatment is associated with clinical benefit, including prolonged stable disease, complete and partial responses, and an excellent safety profile. Studies have shown that 2ME2 is metabolized by conjugation at positions 3 and 17 and oxidation at position 17. To define structure-activity relationships for these positions of 2ME2 and to generate metabolically stable analogues with improved anti-tubulin properties, a series of analogues was generated and three lead analogues were selected, ENMD-1198, ENMD-1200, and ENMD-1237. These molecules showed improved metabolic stability with >65% remaining after 2-h incubation with hepatocytes. Pharmacokinetic studies showed that oral administration of the compounds resulted in increased plasma levels compared with 2ME2. All three analogues bind the colchicine binding site of tubulin, induce G2-M cell cycle arrest and apoptosis, and reduce hypoxia-inducible factor-1α levels. ENMD-1198 and ENMD-1200 showed improved in vitro antiproliferative activities. Significant reductions in tumor volumes compared with vehicle-treated mice were observed in an orthotopic breast carcinoma (MDA-MB-231) xenograft model following daily oral treatment with all compounds (ANOVA, P < 0.05). Significantly improved median survival time was observed with ENMD-1198 and ENMD-1237 (200 mg/kg/d) in a Lewis lung carcinoma metastatic model (P < 0.05). In both tumor models, the high-dose group of ENMD-1198 showed antitumor activity equivalent to that of cyclophosphamide. ENMD-1198 was selected as the lead molecule in this analogue series and is currently in a phase I clinical trial in patients with refractory solid tumors. [Mol Cancer Ther 2008;7(6):1472–82]

Phosphate-binding specificities of monoclonal antibodies against phosphoinositides in liposomes

Monoclonal antibodies against phosphatidylinositol phosphate were produced after injecting a mouse with liposomes containing dimyristoylphosphatidylcholine, cholesterol, phosphatidylinositol phosphate and lipid A. The antibodies raised were IgM (kappa) and their activities were assayed by complement-dependent damage to liposomes lacking lipid A but containing the rest of original immunizing mixture of lipids. Three of the four antibodies selected cross-reacted with liposomes containing phosphatidylinositol instead of phosphatidylinositol phosphate; and two of the antibodies cross-reacted with liposomes containing phosphatidylinositol diphosphate. Each of the antibodies had a phosphate-binding specificity. Each also cross-reacted with liposomes containing sulfogalactosyl ceramide, but not with liposomes containing galactosyl ceramide, or gangliosides or with liposomes containing lipid A but lacking phosphoinositides. Recognition of sulfogalactosyl ceramide probably occurred because the chemical characteristics of the sulfate group were sufficiently similar to those of phosphate to allow recognition by the antibody. The phosphate-binding specificity was further confirmed by inhibition by phosphocholine, inositol hexaphosphate, ATP, AMP and even sodium phosphate, but not by choline or inositol.

Antibodies to phospholipids and liposomes: binding of antibodies to cells.

Binding of two monoclonal anti-liposome antibodies to the surface of cultured murine peritoneal macrophages was investigated by indirect immunofluorescence and enzyme-linked immunosorbent assay. Neither antibody bound to cultures of freshly explanted, nonadherent macrophages, but immunoreactivity was observed following cell adherence to tissue culture plastic. Fluorescent microscopic evaluation revealed heterogeneity in staining patterns of the antibodies on adherent cells. Binding both to viable and fixed adherent macrophages was observed even after a 10,000-fold dilution of antibody. Treatment of adherent macrophage cultures with trypsin increased antibody binding. Further treatment of trypsinized-macrophages with alkaline phosphatase or neuraminidase did not affect antibody binding, but phospholipase D and, to a greater extent, phospholipase C resulted in a marked decrease in cellular binding. The data indicate that antibodies produced against liposomes appear to bind to surface phospholipids of macrophages, but binding can be influenced by the physiological state of the macrophage and overlying cell surface proteins.

Synthesis of 2- and 17-substituted estrone analogs and their antiproliferative structure–activity relationships compared to 2-methoxyestradiol

A novel series of 17-modified and 2,17-modified analogs of 2-methoxyestradiol (2ME2) were synthesized and characterized. These analogs were designed to retain or potentiate the biological activities of 2ME2 and have diminished metabolic liability. The analogs were evaluated for antiproliferative activity against MDA-MB-231 breast tumor cells, antiangiogenic activity in HUVEC, and estrogenic activity on MCF-7 cell proliferation. Several analogs were evaluated for metabolic stability in human liver microsomes and in vivo in a rat cassette dosing model. This study lead to several 17-modified analogs of 2ME2 that have similar or improved antiproliferative and antiangiogenic activity, lack estrogenic properties and have improved metabolic stability compared to 2ME2.

Life-Long Administration of Liposomes and Lipid A in Mice: Effects on Longevity, Antibodies to Liposomes, and Terminal Histopathological Patterns

AbstractLong-term effects of life-long (>2 year) repeated intravenous injections (up to 17) of high doses of liposomes, lipid A, or liposomes containing lipid A were assessed in BALB/c mice. the liposomes contained dipalmitoylphosphatidylcholine, and cholesterol (1/0.75). When compared with mice injected only with normal saline, there were no statistical differences in life spans observed between the different groups. Animals injected with liposomes or liposomes containing lipid A gradually developed “ruffled” fur, but the animals did not appear sick otherwise, and no differences were observed in the mean weights of the animals in the different groups. All of the animals that were tested in each group, including those injected with normal saline, developed IgG antibodies against one or more of nine lipid antigens. the antibodies were detected by a solid-phase enzyme-linked immunosorbent assay (ELisA) and the antigens consisted of either lipid A or one of eight different phospholipids. After 765 days, when...