The angiogenic property of thalidomide reported by D'Amato and colleagues has prompted its clinical evaluation in various solid tumors, including prostate cancer. Previously, we showed that one of the products of cytochrome P450 2C19 isozyme biotransformation of thalidomide, 5'-OH-thalidomide, is responsible for the drug's antiangiogenic activity. Based on the chemical structure of this metabolite, we synthesized 118 analogs of thalidomide and evaluated them using 4 in vitro models to assess activity in the inhibition of angiogenesis (rat aorta model, human saphenous vein model, cultured endothelial cells, and tube formation assay). We identified the most potent of these agents and have patented them. We continue to develop these compounds, which appear to have minimal side effects in initial preclinical toxicology studies. Using a randomized Phase II trial design, we compared weekly docetaxel (30 mg/m2) with or without 200 mg/d of thalidomide to determine whether the combination of thalidomide and docetaxel could produce a sufficiently high clinical response rate to warrant further investigation. A total of 75 patients were enrolled onto this trial, 25 patients in the docetaxel alone arm and 50 patients in the combination arm. Both at the midpoint evaluation and at the conclusion of the trial, the proportion of patients with a greater than 50% decline in PSA was higher in the combination arm (25 of 47 patients, 53%) than in the docetaxel alone arm (9 of 24 patients, 37%). The 18 mo survival was 42.9% in the docetaxel alone group and 68.2% in the combined group. The median overall survival in the docetaxel alone group was 14 mo compared with 28 mo for the combination arm (p=0.11) Thalidomide, Docetaxel and Bevacizumab: Dr. Dahut and I conducted a Phase II trial of thalidomide, docetaxel, prednisone and bevacizumab in chemo-naive CRPC patients. We previously demonstrated that thalidomide appears to add to the activity of docetaxel in metastatic CRPC. Phase II studies combining docetaxel with bevacizumab have shown substantial anti-tumor activity. We hypothesized that the combination docetaxel plus these antiangiogenic drugs with different targets would have substantial clinical activity. To explore safety and efficacy, this was tested in both mouse and patients. Sixty patients with progressive metastatic CRPC received i.v. docetaxel and bevacizumab plus oral thalidomide and prednisone. In the mouse model, combination therapy of docetaxel, bevacizumab, and thalidomide inhibited tumor growth most effectively. In the clinical trial, 90% of patients receiving the combination therapy had PSA declines of greater than or equal to 50%, and 88% achieved a PSA decline of greater than or equal to 30% within the first 3 mo of treatment. The median time to progression was 18.3 mo and the median overall survival was 28.2 mo in this group with a Halabi predicted survival of 14 mo. While toxicities were manageable, all patients developed grade 3/4 neutropenia. The addition of bevacizumab and thalidomide to docetaxel is highly active with manageable toxicities. The estimated median survival is encouraging given the generally poor prognosis of this patient population. These results suggest that definitive clinical trials combining antiangiogenic agents with different mechanisms with docetaxel are warranted to improve treatment outcomes for patients with metastatic CRPC. To maintain the activity of this combination while reducing its associated side effects, we have replaced thalidomide with a structurally similar drug, lenalidomide. In this trial, patients with chemo-naive CRPC will be treated with docetaxel, prednisone, bevacizumab, and lenalidomide. Preliminary study results (28 of a planned 51 pts have been enrolled) showed that the four-agent combination therapy is associated with high response rates, 91.7% in PSA and 78.6% in measurable disease in CRPC pts, with manageable toxicities. In collaboration with Dr. Neil Vargesson, we further investigated the mechanism of action of thalidomide's teratogenic effect. We demonstrated that loss of immature blood vessels is the primary cause of thalidomide-induced teratogenesis and explained its action at the cell biological level. Antiangiogenic but not anti-inflammatory metabolites/analogues of thalidomide induce chick limb defects. Both in vitro and in vivo, outgrowth and remodeling of more mature blood vessels is blocked temporarily, whereas newly formed, rapidly developing, angiogenic vessels are lost. These results explain both the timing and relative tissue specificity of thalidomide embryopathy and have significant implications for its use as a therapeutic agent. Studies are ongoing to evaluate the terotogenicity of the CPS compounds screened in the zebrafish and chick embryo models. A principal mechanism by which cancer cells adapt to the hypoxic microenvironment is through the activity of the transcription factor hypoxia-inducible factor 1 alpha (HIF-1a). HIF-1a expression under hypoxic conditions regulates genes that play key roles in metastasis, angiogenesis, cancer cell metabolism, and resistance. Therefore, the inhibition of transcription driven by HIF (via disrupting the complex that HIF forms with p300, an essential transcriptional coactivator) has the potential for cancer treatment. We have previously shown in our laboratory that several members of the epidithiodiketopiperazine (ETP) family of natural products are able to block the interaction between HIF-1a and p300 by a zinc ejection mechanism. Structure-activity studies using both natural and synthetic ETP derivatives reveal that only the structurally unique ETP core is required and sufficient to block the interaction of HIF-1a and p300. To demonstrate that disruption of the HIF-1a/p300 complex has antiangiogenic effects, we evaluated the activity of selected ETPs, namely chetomin, chaetocin, and gliotoxin in the rat aortic ring model. Chetomin and chaetocin concentrations of 50 nM inhibited approximately 90% of outgrowth, while 500 nM of gliotoxin was needed to achieve a similar effect;these compounds had GI50 of 22, 11, and 175 nM, respectively. We are in the process of determining if the antiangiogenic effects that were observed in our rat aortic ring assays are due to disruption of the HIF/p300 complex through immunoprecipitation assays of the complex from cells in the absence or presence of the ETPs. The active ETPs are being further evaluated for varying potency and specificity in various preclinical studies. Studies are currently underway to identify additional compounds that inhibit the HIF-p300 interaction and involve a high throughput screen using the in vitro fluorescence binding assay developed in our laboratory (composed of a biotinylated synthetic peptide of the C-TAD domain of HIF-1a immobilized on 96-well streptavidin-coated plates and a recombinant GST-tagged protein containing the CH1 domain of p300). We screened a library consisting of 170,000 compounds from the NCI Natural Products Repository that include a collection of pre-fractionation compounds, a selection of compounds known to affect HIF transactivation through unknown mechanisms, and a library of natural compounds. Nine pure compounds have been identified that inhibit the Hif-1a/p300 interaction. Molecular and mass spectrometry studies are being conducted to verify that these compounds can disrupt the HIF-1a/p300 complex and subsequent downstream HIF-mediated signaling. Finally, ex vivo studies will be carried out to evaluate if these compounds have anti-angiogenic effects. We have recently completed testing of LP-261, a novel tubulin targeting anticancer agent in combination with bevacizumab. Oral LP-261 shows antiangiogenic activity and is very effective at inhibiting tumor growth in multiple mouse xenograft models and angiogenesis assays.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIASC006538-18
Application #
8350048
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
18
Fiscal Year
2011
Total Cost
$297,586
Indirect Cost
Name
National Cancer Institute Division of Clinical Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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Ley, Ariel M; Chau, Cindy H; Figg, William D (2015) Structural studies reveal thalidomide's mechanism of action and clinical effects: crystal clear or clearly complexed? Cancer Biol Ther 16:19-20

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