Antiproliferative property of hexadecyloxypropyl 9-[2-(phosphonomethoxy) ethyl] guanine (HDP-PMEG) for unwanted ocular proliferation

Abstract

Purpose: To investigate the safety and inhibitory effects of hexadecyloxypropyl 9-[2-(phosphonomethoxy) ethyl] guanine (HDP-PMEG) on ocular cell proliferation and collagen matrix contraction.

Methods: For the antiproliferation studies, various ocular cell monolayers were exposed to HDP-PMEG, PMEG, 5-fluorouracil (5-FU), and daunorubicin (DNB). For the collagen contraction studies, retinal pigment epithelium (RPE) cells seeded onto type I collagen lattices were exposed for a single 5- or 50-min period to various concentrations of HDP-PMEG or 5-FU. For the cytotoxicity study, trypan blue exclusion tests were performed using a human Müller cell line. Cytotoxicity was determined up to 4 days after treatment.

Results: The proliferation of RPE cells, scleral fibroblasts, vessel endothelial cells, and ocular melanoma cells can all be significantly inhibited by HDP-PMEG. Its inhibitory effects on those cells were uniformly stronger than that of 5-FU. Contraction of the collagen matrix containing RPE cells was significantly inhibited by HDP-PMEG and by 5-FU at concentrations of 20 µM and 2,000 µM, respectively, as compared with controls (p<0.05). The safety profile of HDP-PMEG was significantly better than 5-FU and daunorubicin. The ocular therapeutic index is 1,100 for HDP-PMEG, 17.2 for 5-FU, and 1.25 for daunorubicin.

Conclusions: HDP-PMEG possesses a significant inhibitory effect on the proliferation of RPE, retinal glial cells, scleral fibroblasts, and ocular melanoma cells. HDP-PMEG is also genotoxic and may be used as a single short application for the modulation of unwanted ocular proliferation.

Figure 1

Chemical structure of PMEG and HDP-PMEG.

Figure 2

MTS cell proliferation assay. Upper panel: proliferation assay on ARPE19 cell. Center panel: proliferation assay on Muller cell. Bottom panel: proliferation on EA-HY926 endothelium cell.

Figure 3

MTS cell proliferation assay to demonstrate the inhibitory effect on proliferation of the three melanoma cell lines: M23 cell (Upper panel), OCM1 cell (Center panel), and SP6.5 cell (Bottom panel). For M23 and SP6.5 cells, HDP-PMEG demonstrated a similar inhibitory effect to 5-FU while HDP-PMEG had a consistent stronger inhibitory effect than 5-FU on the OCM1 cell. Daunorubicin had the strongest inhibitory effect on all the three cell lines.

Figure 4

Cytotoxicity assay on Müller cell. A: The percentage of trypan blue positive cells (y-axis) was plotted against the concentration of the test compounds used (x-axis). The concentration unit was micro molar and the open box represented a 95% confidence limit. 5-FU at 100 µM that was the highest concentration tested, caused 20% death of the cell culture, which was equivalent to HDP-PMEG at 11 µM or DNR at 0.1 µM. B: Exemplary images from the Müller cell cytotoxicity study: the upper left panel showing a counting field from the control in which 31 cells were stained by trypan blue out of a total of 435 cells, yielding a 7% trypan blue positive rate. The rest of the three fields had a trypan blue positive rate of 19% (38/198) for 10 µM HDP-PMEG, 18% (46/251) for 100 µM 5-FU, and 18% (49/274) for 0.1 µM daunorubicin (DNR).

Figure 5

The ARPE19 cell proliferation inhibitory effect from single short-term exposure of HDP-PMEG and 5-FU. A: Following a 50-min contact, 2 µM and 6.32 µM HDP-PMEG inhibited cell proliferation by 50% and 75%, respectively, which were equivalent to 200 µM and 2,000 µM of 5-FU. The 5-min exposure also induced significant proliferation inhibition. However, the inhibition was consistently less than that from 50-min exposure cross all concentrations. The mark on the top of each bar represents the standard deviation. B: The Müller cell proliferation inhibitory effect from a single short-term exposure of HDP-PMEG and 5-FU. Both 5- and 50-min exposure induced dose dependent inhibition by 5-FU and HDP-PMEG. In a 50-min exposure, 2 µM HDP-PMEG induced a similar growth inhibition as that observed by 200 µM 5-FU. C: The anterior scleral fibroblast proliferation inhibitory effect from a single short-term exposure of HDP-PMEG and 5-FU. For the anterior scleral fibroblasts, a 20 µM HDP-PMEG 5-min exposure induced a 65% inhibition while even 20 mM 5-FU did not achieve the similar magnitude growth inhibition.