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.

Purpose: Head and neck squamous cell carcinomas have been reported to overexpress hypoxia-inducible factor (HIF)-1␣, 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-1␣ 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 mol/L range, including induction of G 2 -M blockade, caspase-3/7 activation, and apoptosis at 48 hours. 2ME2 resulted in decreased nuclear HIF-1␣-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-1␣, because HIF-1␣ 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 containing antimonial compounds trapped in the aqueous phase were tested in the treatment of experimental leishmaniasis. The rationale of this approach was based on the hypothesis that the liposomes and the parasite are taken up by the same cell, the reticuloendothelial cell, and we present electron microscopic evidence that supports this hypothesis. Suppression of leishmaniasis was quantified by determining the total number of parasites per liver from impression smears. When two antimonials, meglumine antimoniate and sodium stibogluconate, were encapsulated within lipo somes, each was more than 700 times more active compared to either of the free (unencapsulated) drugs. After infection, if untreated, all of the hamsters eventually would die from the disease. Liposome-encapsulated meglumine antimoniate was about 330-640 times more effective in causing a drop in the death rate than was the free antimonial. The efficacy of treatment was influenced by the lipid composition and charge of the liposomes. For example, positively charged liposomes containing egg phosphatidylcholine were much less effective than negatively charged ones. In contrast, positively and negatively charged sphingomyelin liposomes were equally effective. Liposomes containing phosphatidylserine (which were negatively charged, but also had a much higher charge density) were among the less-effective preparations. Among those tested, the most consistently efficacious liposomes contained highly saturated long-chain phospholipids (eg., dipalmitoyl phosphatidylcholine), cholesterol, and a negative charge.We conclude that liposomes may be useful as carriers of drugs to treat infectious diseases involving the reticuloendothelial system. The toxicities of antimony are very similar to those of arsenic. Encapsulation of antimonial drugs and reduction of the dose required for effective therapy should minimize such systemic toxicities as acute cardiomyopathy and toxic nephritis.

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 hep-

Cholesterol-dependent complement activation has been proposed as a factor that might influence the pathogenesis of atherosclerosis. Although antibodies to cholesterol conjugates have been reported, cholesterol is widely regarded as a poorly immunogenic substance. Monoclonal IgM complement-fixing antibodies to cholesterol were obtained in the present study after immunizing mice with liposomes containing high amounts of cholesterol (71 mol % relative to phosphatidylcholine) and lipid A as an adjuvant.Clones were selected for the ability of secreted antibodies to react with liposomes containing 71% cholesterol but not with liposomes containing 43% cholesterol. The antibodies also reacted with crystalline cholesterol in a solid-phase enzymelinked immunosorbent assay. Binding of monoclonal antibodies to the surface of crystalline cholesterol was demonstrated by electron microscopy by utilizing a second antibody (antilgM) labeled with colloidal gold. The immunization period required to induce monoclonal antibodies was very short (3 days) and a high fraction of the hybrid cells (at least 70%) were secreting detectable antibodies to cholesterol. The results demonstrate that cholesterol can be a highly immunogenic molecule and that complement-flxing antibodies to cholesterol can be readily obtained.Cholesterol has been widely studied as a fundamental and ubiquitous constituent of cell membranes and lipoproteins (1,2). Despite occasional reports that conjugates of cholesterol can be immunogenic (3)(4)(5), because of its widespread distribution and important biological roles cholesterol has generally been assumed to be a nonimmunogenic or poorly immunogenic molecule. However, since 1977 several laboratories have reported activation of the classical and alternative pathways of complement by cholesterol (6-11). The exact mechanism (or mechanisms) of complement activation by cholesterol has not been determined. In one case human IgG antibodies to cholesterol were implicated in the activation of the classical pathway (9). On the other hand, it has been proposed that nonimmune activation of the alternative pathway of rabbit complement (7) or human complement (8, 9) also occurs. The issue of the immunogenicity of cholesterol is an important one, since antibodies to cholesterol have been reported to protect against induced atherosclerosis in rabbits (4). On the other hand, activation of the classical pathway of complement by cholesterol has been proposed as a possible mechanism in the pathogenesis of atherosclerosis (6,9,11).In previous studies we induced antibodies to the phospholipid constituents of lipid model membranes (liposomes) (reviewed in ref. 12). Production of antibodies to liposomes was achieved by utilizing lipid A as a powerful adjuvant in the liposomal lipid bilayer. In the present work we used lipid A as an adjuvant to induce antibodies to liposomes containing high concentrations of cholesterol. We found that cholesterol is an excellent immunogen and that murine monoclonal antibodies to cholesterol are eas...

Tissue factor pathway inhibitor (TFPI) is the major physiologic inhibitor of the extrinsic coagulation pathway. We have previously shown that TFPI is also a potent inhibitor of endothelial proliferation in vitro and of primary and metastatic tumor growth in vivo. Surprisingly, the antitumor activity of TFPI was demonstrated to be independent of its anticoagulant activity, suggesting a possible nonhemostatic mechanism of action for TFPI in these models. This antitumor mechanism may involve the very low density lipoprotein (VLDL) receptor because the in vitro antiproliferative activity of TFPI is mediated through interaction with the VLDL receptor. In the current study, we identify a 23-amino acid fragment of TFPI (TFPIc23) localized to the C-terminus, which mediates binding to the VLDL receptor. The TFPIc23 peptide inhibits endothelial cell proliferation through an apoptotic mechanism and blocks vessel outgrowth in the in vitro assays, and this activity is mediated through interaction with the VLDL receptor. In vivo, this peptide potently inhibits angiogenesis in Matrigel and chick chorioallantoic membrane models and also inhibits metastatic tumor growth. Our data demonstrate that this VLDL receptor-binding fragment of the TFPI molecule has apoptotic, antiangiogenic, and antitumor activity and suggests a possible mechanism whereby TFPI can regulate angiogenesis and tumor growth independently of its anticoagulant activity. (Blood. 2004;103: 3374-3380)

Administration of liposome-encapsulated ribavirin to mice led to ribavirin concentrations in the liver, the primary site of Rift Valley fever virus proliferation, that were fivefold greater than those attained with the same doses of free ribavirin. Liposomal ribavirin given at a dose of either 25 or 50 mg of drug per kg of body weight protected mice against a rapidly lethal high-titer challenge with Rift Valley fever virus, whereas similar doses of free drug or empty liposomes had no detectable benefit. Hence, tissue targeting of ribavirin with liposomes substantially increased the therapeutic index by increasing the efficacy of the treatment. By using liposomes as drug carriers, a nontoxic, low-dose regimen of ribavirin had a therapeutic effect that was comparable to that achieved with higher but potentially more toxic doses of free ribavirin.

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