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. 2012 Feb 1;72(3):716-25.
doi: 10.1158/0008-5472.CAN-10-2873. Epub 2011 Dec 8.

Metronomic dosing of BH3 mimetic small molecule yields robust antiangiogenic and antitumor effects

Atsushi Imai  1 , Benjamin D Zeitlin, Fernanda Visioli, Zhihong Dong, Zhaocheng Zhang, Sudha Krishnamurthy, Emily Light, Frank Worden, Shaomeng Wang, Jacques E Nör
Affiliations

Metronomic dosing of BH3 mimetic small molecule yields robust antiangiogenic and antitumor effects

Atsushi Imai et al. Cancer Res. .

Abstract (V体育官网入口)

Bcl-2 is an antiapoptotic protein that has also been found to function as a proangiogenic signaling molecule. Improvements in antiangiogenic therapy can be engendered by metronomic dosing. Thus, we hypothesized that BH3-mimetic drugs that antagonize Bcl-2 family proteins may exert a greater efficacy when dosed metronomically. To examine this hypothesis, we employed AT101, an orally available and well-tolerated BH3-mimetic drug that has been established as effective. In a mouse xenograft model of human squamous cell carcinomas (SCC) that includes a humanized vasculature, we explored the effects of docetaxel in combination with either daily (metronomic) or weekly (bolus) doses of AT101. In addition, we explored the effect of single or combination therapy on angiogenesis and survival of endothelial or SCC cells in vitro VSports手机版. Metronomic AT101 therapy increased mouse survival, decreased tumor mitotic index, and decreased tumor microvessel density, compared with bolus therapy. Therapeutic potentiation was achieved by similar overall drug exposure and without altering systemic toxicities. Combinations of AT101 and docetaxel produced additive toxicity in both endothelial and SCC tumor cells. Notably, subapoptotic concentrations of AT101 potently inhibited the angiogenic potential of endothelial cells. Taken together, our findings unveil the efficacious benefits that can be achieved by metronomic delivery of BH3-mimetic drugs, in particular suggesting that SCC patients with might benefit from low-dose continuous administration of these drugs. .

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Conflict of interest statement (VSports app下载)

Disclosure of Potential Conflicts of Interest: Shaomeng Wang owns stocks, stock options and serves as a consultant for Ascenta Therapeutics that has licensed technologies related to AT101 from the University of Michigan.

"V体育ios版" Figures

Figure 1
Figure 1
Metronomic AT101 enhances the time to failure of xenograft head and neck tumors. To generate human xenografts vascularized with human vessels, mice received a scaffold seeded with human head and neck squamous cell carcinoma cells (OSCC3) and human endothelial cells. When tumors reached 200 mm3, mice were randomized into the different treatment regimens (vehicle, n=8; taxotere, n=7; weekly AT101+taxotere, n=8; daily AT101+taxotere, n=8). Daily administration of low dose AT101 (10 mg/kg) was designated as “metronomic” regimen, while weekly administration of high dose AT101 (70 mg/kg) was designated as “bolus” regimen. In both cases, AT101 was delivered via oral gavage and treatment was performed for 3 weeks. In all conditions, 5 mg/kg taxotere (TXT) were administered weekly via intraperitoneal injection. A, Kaplan-Meier analysis using as criterion for failure a tumor volume of 2,000 mm3. P-value indicates a significant difference across the four groups (p<0.0001). Asterisk (*) depicts a statistical difference between weekly AT101+taxotere and daily AT101+taxotere (p=0.02). B, graph depicting tumor volume from the first day of treatment through the day when the average tumor volume per group reached 1,000 mm3. C, distribution of Wilcoxon scores for log2 tumor volume at 13 days of treatment, i.e. the day in which the first mouse of the control group had to be euthanized because the tumor reached the cutoff size of 2,000 mm3. P-value indicates a significant difference across the four groups (p<0.0001). Different letters depict statistically significant differences based on pairwise comparisons (p<0.05). D, graph depicting average body weight during treatment, normalized against pre-treatment weight.
Figure 2
Figure 2
Metronomic AT101 lowers the mitotic index and tumor microvessel density. Paraffin embedded tissue sections were prepared from tumors evaluated in Figure 1. A, representative images of histological sections stained with hematoxilin/eosin (top row, ×100; middle row, ×400). Representative images (bottom row, ×200) of histological sections immunostained for factor VIII (red stain) to identify blood vessels and counterstained with hematoxylin. B, distribution of Wilcoxon scores for average mitotic cells per mm2 (mitotic index), as determined by a trained pathologist blinded for experimental conditions. C, distribution of Wilcoxon scores for average tumor microvessel density assessed in 6 high power fields per tumor. P-value indicates a significantly difference across the four groups (p=0.0007, p=0.0042, respectively). Different letters depict statistically significant differences based on pairwise comparisons (p<0.05).
Figure 3
Figure 3
Cytotoxic profile of AT101 and taxotere in endothelial cells and head and neck tumor cells. The cytotoxicity was determined by the SRB assay. A to D, cytotoxicity of AT101 and taxotere (TXT) on human dermal microvascular endothelial cells (HDMEC) and head and neck squamous cell carcinoma cells (OSCC3, UM-SCC-74A, UM-SCC-17B). Cells were exposed to AT101 or TXT for 72 hours. Data were normalized against vehicle control and initial plating density. Experiments were done in triplicate wells per condition. Each graph is representative of three independent experiments.
Figure 4
Figure 4
Effect of AT101 and taxotere drug combination on endothelial cells and head and neck tumor cells. The cytotoxicity was determined by the SRB assay. A to D, cells were exposed to AT101 or taxotere (TXT) for 72 hours. The concentrations selected for this study were the IC25, IC50 and IC75 for each drug in each cell line. Combinatorial Index (CI) was calculated for each experimental condition. Asterisk (*) depicts synergistic effect (CI<0.9), while a plus sign (+) depicts additive effect (0.9≤CI≤1.1). Data were normalized against vehicle control and initial plating density. Experiments were done in triplicate wells per condition. Each graph is representative of three independent experiments.
Figure 5
Figure 5
Effect of drug sequence on endothelial cells and head and neck tumor cells. A to D, cells were exposed to the IC50 concentration of each drug, and cytotoxicity was determined by the SRB assay. Cells were pre-treated either with AT101 or with TXT for 24 hours. Then, cells were exposed to AT101 or taxotere (TXT) for additional 48 or 72 hours. Alternatively, treatment was performed with both drugs administered at the same time, or performed with single-drug as controls. Asterisk (*) depicts statistically significant differences at p<0.01. Data were normalized against initial plating density. Experiments were done in triplicate wells per condition. Each graph is representative of three independent experiments.
Figure 6
Figure 6
Effect of metronomic versus bolus AT101 on the angiogenic potential of human endothelial cells. The effect of AT101 on the angiogenic potential was evaluated in primary human dermal microvascular endothelial cells (HDMEC) plated in 3-D collagen matrices. A, graph depicting the number of capillary sprouts in VEGF-treated HDMEC exposed to AT101 for 4 days. AT101 was used at the following concentrations: 0.1×IC50 (0.08 µM), IC50 (0.8 µM), 10×IC50 (8 µM). In the metronomic group, 1/4 of the IC50 was administered daily (from day 0 to 3); while in the bolus treatment group, the full dose was administered on day 0. The total amount of drug was the same in the metronomic and bolus treatment group. B, graph depicting the number of sprouts at the end of the experimental period. Different letters depict statistically significant differences at p<0.05. C, representative photomicrographs of capillary sprouts (×150) at the end of the experimental period. Results were normalized against initial number of sprouts. Experiments were done in triplicate wells per condition. Data is representative of three independent experiments.
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