An orally delivered small-molecule formulation with antiangiogenic and anticancer activity

Abstract

Targeting angiogenesis, the formation of blood vessels, is an important modality for cancer therapy. TNP-470, a fumagillin analog, is among the most potent and broad-spectrum angiogenesis inhibitors. However, a major clinical limitation is its poor oral availability and short half-life, necessitating frequent, continuous parenteral administration. We have addressed these issues and report an oral formulation of TNP-470, named Lodamin. TNP-470 was conjugated to monomethoxy-polyethylene glycol-polylactic acid to form nanopolymeric micelles. This conjugate can be absorbed by the intestine and selectively accumulates in tumors. Lodamin significantly inhibits tumor growth, without causing neurological impairment in tumor-bearing mice. Using the oral route of administration, it first reaches the liver, making it especially efficient in preventing the development of liver metastasis in mice. We show that Lodamin is an oral nontoxic antiangiogenic drug that can be chronically administered for cancer therapy or metastasis prevention.

Figure 1

Lodamin synthesis and characterization. (a) Diagram of the conjugation reaction between TNP-470 and modified mPEG-PLA. 1. The reaction between succinated mPEG-PLA and ethylendiamine that results in an amine-terminated polymer. 2. The reaction between the amine-containing polymer and the terminal chlorine of TNP-470. The conjugate is then dialyzed against water in an excess of TNP-470 to form polymeric micelles. (b) A typical DLS measurement of Lodamin, the graph shows size distribution of the polymeric-micelles in water. (c) TEM images of Lodamin dispersed in water, the spherical structure of the micelles are shown at different time points after incubation in water. Scale bar, 10 nm. (d) TNP-470 release from Lodamin during a 30-d period as determined by HPLC; micelles were incubated in gastric fluid pH = 1.2 or PBS pH = 7.4 and analysis of the released TNP-470 was done in duplicate. The results are presented as means ± s.d. mPEG-PLA, methoxy-polyethylene glycol–polylactic acid; suc, succinated; EDC, ethyl(diethylaminopropyl) carbodiimide; NHS, N-hydroxysuccinimide; en, ethylenamine; d.d.w., double-distilled water; DMF, dimethylformamide.

Figure 2

Effect of Lodamin on angiogenesis and cell uptake of the drug. (a) Left: Confocal images show HUVEC uptake of polymeric micelles labeled with 6-coumarin after 20 min and 7-h incubation time periods. Right: Live HUVECs imaged 1 h after the addition of labeled micelles to cell medium (15 μg/ml, in green). LysoTracker Red detecting endosomes and lysosomes is shown in red. Overlay between micelles and endosomes/lysosomes is represented in yellow/orange color. Scale bars, 5 μm. (b) Uptake of rhodamine-labeled mPEG-PLA micelles by HUVECs was evaluated by FACS analysis. A viable cell gate was established based on forward and side scatter (FSC and SSC), and another gate was set for SSC/FL4_high-measurement of HUVECs containing mPEG-PLA-rhodamine micelles. Graphs of 0 min, 20 min and 24 hr of incubation with micelles are shown. Incubation after 2, 4 and 7 h showed a similar pattern as after 24 h of incubation (not shown). (c) HUVEC growth and proliferation. Upper panel: inhibition of HUVEC proliferation by different concentrations of Lodamin 50–1,000 nM TNP-470 equivalent. Empty polymeric micelles were also added as a control (n = 8, *P < 0.0005). Lower panel: HUVEC growth curve of cells treated every other day with Lodamin (60 nM TNP-470 equivalent), untreated cells and vehicle-treated cells. (d) Corneal micropocket assay: representative experiments of the corneal micropocket assay. Newly formed blood vessels are growing toward the bFGF pellet (in white); note the inhibition of angiogenesis in Lodamin-treated mouse (15 mg/kg per day) with respect to control. Lower panel shows the quantification of neovascularization area in the cornea (n = 10, mean ± s.d.).

Figure 3

Intestinal absorption and body distribution studies. (a) Intestinal absorption of mPEG-PLA micelles. Left, above: A histological section of gut epithelial cells of Lodamin-treated mouse observed with TEM. Microvilli (MV) structures and endosomes loaded with polymeric micelles (E, arrows) are detected. Scale bar, 500 nm (left) and 200 nm (right). Below: Confocal microscopy image. Note that the polymeric micelles can be detected in the lamina propria in the vicinity of blood vessels. The actin filaments were stained with phalloidin-FITC (green), nuclei were labeled with DAPI (blue) and mPEG-PLA-rhodamine micelles were detected in red. Scale bars, 5 μm. (b) Fluorescent signal of tissue extracts and in serum. Mice (n = 3) were given a single dose of oral 6-coumarin–labeled polymeric micelles (150 μl, 30 mg/ml). Left: the graph shows the values of the three different mice, autofluorescence was omitted by subtracting the fluorescent signal of tissue extracts from an untreated mouse. The percent of labeled cells was measured for each organ. Right: levels of fluorescent signals in mouse serum as measured after different time points after oral administration. The results are presented as the concentration of micelles calculated by standard calibration curve. (c) The percentage of FL2^high-positive cells is shown as isolated from the designated organs (ratio of numbers FL2^high cells of treated tumors to these of control mouse). (d) FACS analysis graphs of single-cell suspensions from three representative organs (tumor, liver, brain) taken from a mouse bearing Lewis lung carcinoma after controlled enzymatic degradation. The FL2^high represents those cells that contain the mPEG-PLA-rhodamine micelles.

Figure 4

Lodamin inhibits primary tumor growth without causing neurotoxicity. (a) Effect of free or conjugated TNP-470 on established Lewis lung carcinoma tumors: effect of 30 mg/kg every other day of free TNP-470 given orally, compared to equivalent dose of Lodamin or water (n = 5 mice per group, *P < 0.05). (b) Lewis lung carcinoma volume during 18 d of different frequencies and doses of Lodamin: 30 mg/kg every other day (q.o.d.), 15 mg/kg every other day, 15 mg/kg every day (q.d.) and water by gavage (n = 5 mice per group, *P < 0.05. (c) Evaluation of the effect of the vehicle, empty mPEG-PLA micelles, on Lewis lung carcinoma (n = 5 mice per group, *P < 0.05). (d) Effect of Lodamin given at a dose of 15 mg/kg every day on B16/F10 murine melanoma tumor in C57Bl/6J, water was given as control (n = 5 mice per group, *P < 0.05). (e) Representative Lewis lung carcinoma and B16/F10 tumors removed from mice at day 18 after treatment with oral Lodamin at 30 mg/kg every other day and 15 mg/kg every day, respectively, and from control untreated mice. (f) Neurotoxicity evaluation of Lodamin-treated mice (10 d, 30 mg/kg every other day) compared to mice treated with subcutaneous (30 mg/kg every other day) free TNP-470 or water given by gavage. Balance beam test was quantified by foot-slip errors and the numbers of slips per meter are presented (n = 4–5 mice per group). *P < 0.05, **P < 0.01, ***P < 0.0001 (results are mean ± s.e.m.).

Figure 5

Lodamin effect on tumor structure, cell proliferation, angiogenesis and apoptosis. Lewis lung carcinoma (LLC) tumors were removed from Lodamin-treated or untreated mice and sectioned. Tissues were stained with H&E to detect tissue morphology. Immunostaining with anti-CD31 was used to detect microvessels and anti-Ki-67 nuclear antigen for cell proliferation. TUNEL staining was used for the detection of apoptotic cells in lower panels. Vessels are detected in green, apoptotic cells are detected in red by TUNEL and cell nuclei are in blue (DAPI). Sections were counterstained with eosin (nuclei). Arrows indicate vessel structures. Scale bars, 15 μm.

Figure 6

Lodamin inhibits liver metastasis in mice injected with B16/F10 cells into their spleen. (a) Livers removed from Lodamin-treated or untreated mice, 20 d after cell injection. Control livers were enlarged with widespread macroscopic malignant nodules and extensive cirrhosis. (b) Histology of liver tissues. Livers removed from Lodamin-treated or untreated mice stained with H&E (upper panel) or reacted with anti-mouse melanoma antibody (lower panel). B16/F10 melanoma cells are detected by positive DAB staining (brown). (c) Spleens removed from Lodamin-treated or untreated mice. Control spleens had large masses compared to treated mice with normal spleen morphology. (d) Survival curve of treated versus control mice (n = 7). Treatment started at day three (TX) after cell injection (arrow).