Inhibition of Intraocular Tumor Growth by Topical Application of the Angiostatic Steroid Anecortave Acetate

Abstract

purpose. This study examined the effect of an angiostatic agent on the growth of a highly vascularized intraocular tumor.

methods. A murine uveal melanoma cell line (99E1) was transplanted intracamerally into athymic nude BALB/c mice. Mice were treated topically three times per day beginning on the day of tumor transplantation and continuing through day 28. Groups included (a) 1% anecortave acetate, (b) vehicle control, or (c) no treatment. Tumor growth was scored clinically according to the volume of anterior chamber occupied by tumor. Intraocular tumor weights were determined on days 10, 14, 21, and 28. The effect of the test agents on tumor cell proliferation was examined in vitro by [³H]thymidine incorporation.

results. Tumors grew progressively in untreated mice and mice treated with the vehicle; tumors filled the entire eye by day 20 and frequently perforated the globe by day 21. By contrast, tumors treated with anecortave acetate grew significantly slower (P < 0.025) and did not perforate the eye. On days 21 and 28 the net tumor weight of the AL-3789–treated animals was 40% to 30% of controls (P < 0.05). Tumor inhibition was presumably due to the angiostatic properties of anecortave acetate because the compound did not affect tumor cell proliferation in vitro.

conclusions. The topical ocular administration of anecortave acetate restricted the growth of a highly vascularized angiogenic intraocular tumor.

Angiogenesis, the process in which new capillaries sprout from existing vessels, is crucial for embryonic development, growth, tissue repair, and certain disease processes. ¹ The role of angiogenesis in the growth and metastasis of solid tumors is well recognized. ¹ Recently, considerable effort has focused on the inhibition of angiogenesis as a strategy for controlling the growth and metastasis of various solid tumors. ¹ A variety of agents have been proposed for inhibiting angiogenesis, including antagonists of vascular endothelial growth factor (VEGF) or VEGF receptors, fumagillin, α-interferon, and compounds that interfere with adhesion to cell matrices. ²  

The angiostatic steroids are an important class of angiostatic agents. These steroidal compounds were first described as being angiostatic in the chicken embryo chorioallantoic membrane (CAM) model of neovascularization, and angiostatic activity appeared to be independent of steroid hormone activity. ³ A new angiostatic steroid, anecortave acetate (AL-3789), recently has been demonstrated to have significant antiangiogenic activity in a wide variety of neovascular models, including the chick embryo CAM, ⁴ lipopolysaccharide-induced corneal neovascularization in the rabbit, ⁵ rat pup hypoxia-induced retinal neovascularization, ⁶ and retinopathy of prematurity in the kitten (Phelps DL, Collier RJ, and Clark AF, unpublished observation, November 1993). 

The purpose of the present study was to determine whether topical ocular administration of anecortave acetate could inhibit the growth of an intraocular tumor. The tumor model was generated using the 99E1 transgenic tumor cell line that has been shown to be very rapidly growing and highly neovascular. ^{7 8}  

Methods

Mice

Female athymic nude BALB/c (H-2^d) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and were incorporated into experiments when 8 to 10 weeks of age. The use of animals conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Tumor Cell Line

The 99E1 tumor cell line was derived from a choroidal/retinal pigmented epithelial ocular tumor that arose in a transgenic FVB/N mouse bearing the SV40 oncogene. ⁷ This tumor cell line expresses SV40 T antigen, as well as melanoma-associated antigens. ^{7 8} Tumor cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO BRL, Grand Island, NY) containing 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT), 1% l-glutamine (JRH Biosciences, Lenexa, KS), 1% sodium pyruvate, 1% vitamin solution, and 1% antibiotic-antimycotic solution (Biowittaker, Walkersville, MD). This tumor cell line was chosen for this study because of its similarity to human uveal melanoma and because it arose by in situ transformation within the uveal tract/retinal pigment epithelium of an FVB/n mouse. ^{7 8}  

Intracameral Transplantation

A modified quantitative technique for the intracameral (IC) transplantation of precise numbers of tumor cells into the anterior segment of the mouse eye has been described previously. ⁸ Mice were deeply anesthetized with 0.66 mg ketamine hydrochloride (Vetalar; Parke, Davis, and Co., Detroit, MI) given intramuscularly. Tumor cells (10⁵/5 μl) were inoculated into the anterior chamber, using a 1.0-ml Hamilton syringe fitted with a 35-gauge glass needle. 

Preparation and Topical Administration of Anecortave Acetate

Anecortave acetate (AL-3789)[ 4,9(11)-pregnadien-11β,17α,21-triol-3,20-dione-21-acetate] was prepared as a proprietary 1% nonsettling suspension. Control animals received the same vehicle. Groups of animals (n = 5 to 10 mice/group) were treated three times a day with 4 μl of 1% anecortave acetate or with vehicle while a third group remained untreated. 

Assessment of Tumor Mass

Eyes were examined two to three times per week using an operating microscope and the tumor growth scored based on the relative tumor mass that occupied the anterior chamber. ⁸ Tumor-containing eyes and the contralateral, normal eyes were enucleated at necropsy and weighed. The difference between the weight of the tumor-containing eye and the contralateral normal eye was considered to be an approximation of the tumor mass for each mouse. 

Assay for In Vitro Tumor Cell deacetylated

The effect of anecortave acetate and its deacetylated metabolite on 99E1 tumor cell proliferation was assessed in vitro. 99E1 cells were suspended in complete DMEM (10⁵ cells/ml), and 100 μl was added to each well in 96-well microtiter plates. One hundred microliters of DMEM containing anecortave acetate or its deacetylated metabolite (AL-4940) at 10⁻⁶ M or 10⁻⁷ M concentrations were added to each well. Cultures were incubated at 37°C for 24, 48, and 72 hours. During the final 18 hours of incubation, 1 μCi of [³H]thymidine (Amersham Co., Arlington Heights, IL) was added to each well. Cells were harvested onto filter papers, using a MASH II cell harvester (MA Bioproducts, Walkersville, MD), and the incorporation of[ ³H]thymidine was determined by liquid scintillation counting on a Beckman scintillation counter (Beckman Instruments, Inc., Irvine, CA). 

Statistics

Longitudinal growth models were fit to the clinical score data plotted as the mean clinical score versus time. The slopes of the lines were compared by linear regression analysis. Statistical significance in tumor weights between experimental and control groups was determined by Student’s t-test. Significance was assumed when P < 0.05. 

Results

As in previous studies, ⁸ 99E1 melanomas grew rapidly after IC transplantation. Intraocular tumors grew in all mice, although the extent of tumor growth varied between the treated and untreated groups. Tumor growth patterns in mice treated with the vehicle did not differ significantly from the untreated control group. Tumors perforated the globe of the eyes between days 21 to 28 in approximately one half (4/9) of the mice in the untreated and vehicle groups. However, topical application of anecortave acetate resulted in a marked inhibition of clinically assessed tumor growth (Figs. 1 2) . The growth rate of tumors in the control (vehicle and untreated) animals was significantly greater that the tumor growth rate in the anecortave acetate–treated group (P < 0.025). Tumors did not perforate the globe in any of the treated mice. These clinical observations were confirmed by weighing the tumor-containing eyes. Anecortave acetate treatment produced 44%, 40%, 61%, and 70% reductions in the tumor weights of the tumor-containing eyes on days 10, 14, 21, and 28 compared to their respective control groups (P < 0.05 for days 10, 21, and 28) (Fig. 3) . Tumor weights (mean ± SEM in milligrams) for the control group were 6.26 ± 1.07 (n = 10) for day 10, 9.04 ± 2.55 (n = 10) for day 14, 43.03 ± 8.67 (n = 11) for day 21, and 110.7 ± 29.6 (n = 9) for day 21. Tumor weights in the anecortave acetate group were 3.49 ± 1.11 (n = 10) for day 10, 5.46 ± 3.20 (n = 5) for day 14, 16.72 ± 6.30 (n = 5) for day 21, and 32.62 ± 13.66 (n = 5) for day 28. The clinical scores appeared to underestimate the efficacy of anecortave acetate compared to the tumor weights most likely because the clinical scores were based on visual inspection of tumor size in the anterior segment and did not account for tumor growth in the posterior segment. 

The inhibition of intraocular tumor growth was presumably due to the potent angiostatic properties of anecortave acetate although it was theoretically possible that this compound was either directly cytotoxic to tumor cells or inhibited tumor cell proliferation. However, the results from in vitro assays indicated that neither the active nor the deacetylated form of anecortave acetate affected the viability or the proliferation of 99E1 tumor cells over a 72-hour period (Fig. 4) . 

Discussion

The growth of solid tumors is limited by the tumor’s ability to obtain a nutritive source via stimulation of new blood vessel growth. Injection of 99E1 tumor cells into the anterior chamber of nude mice leads to a rapidly proliferating and highly neovascular tumor. Topical ocular administration of the angiostatic steroid anecortave acetate beginning at the time of injection of tumor cells led to a significant decrease (40%–70% inhibition) in tumor growth rate over the 4-week period of treatment. Neither anecortave acetate nor AL-4940 (the deacetylated metabolite) affected tumor cell proliferation in vitro, suggesting that this agent inhibited tumor growth by its angiostatic action. 

Anecortave acetate has been shown to have considerable angiostatic activity in several different neovascularization model systems. It is one of the more potent angiostatic steroids in the chicken embryo CAM model of neovascularization. ⁴ Topical ocular administration of anecortave acetate four times per day or two times per day almost totally inhibited LPS-induced corneal neovascularization in rabbits. ⁵ A single intravitreal injection of anecortave acetate into rat pups significantly inhibited hypoxia-induced retinopathy. ⁶ Likewise, a single intravitreal injection of AL-4940 into the eyes of kittens in a retinopathy of prematurity model caused a 50% reduction in retinal neovascularization compared to vehicle injected eyes (Phelps DL, Collier RJ, and Clark AF, unpublished observation November 1993). 

Unlike many other angiostatic agents, it appears that anecortave acetate is angiostatic in a wide variety of neovascularization models, independent of the species, the tissue undergoing angiogenesis, and the initial angiogenic signal. Anecortave acetate is a 21-acetate ester that is deacetylated rapidly in the eye and blood. It displays effective ocular penetration when applied topically to the eye and therefore offers good bioavailability. ⁴ Other angiostatic steroids, such as medroxyprogesterone acetate, have been reported to inhibit urokinase plasminogen activator (uPA) activity ^{9 10} and upregulate plasminogen activator inhibitor (PAI) expression. ¹⁰ One of the rate-limiting steps in angiogenesis consists of an angiogenic signal-mediated stimulation of vascular endothelial cell (VEC) uPA activity to enable the VECs to break through the vessel basement membrane and migrate through interstitial tissue toward the angiogenic signal (i.e., the tumor). At least a portion of anecortave acetate’s angiostatic activity appears to be due to the inhibition of vascular endothelial cell uPA and stromelysin expression (DeFaller JM, McNatt LG, and Clark AF, unpublished observation), as well as the upregulation of PAI expression in the retinas of rats in a model of retinopathy of prematurity. ¹¹  

There are a number of ocular diseases in which neovascularization plays a major role in the loss of vision. It is our hope that new angiostatic agents will be discovered that significantly interfere with ocular neovascularization and thereby preserve vision. Although anecortave acetate is active in a variety of neovascular animal models, including the inhibition of intraocular tumor growth shown in the present study, further evaluation is required to determine whether it will be useful for preserving vision in humans. 

 Reprint requests: Abbot F. Clark, Alcon Laboratories, Inc., R2-41 6201 South Freeway, Fort Worth, TX 76134-2099.

 Submitted for publication July 9, 1998; revised February 11, 1999; accepted April 13, 1999.

 Proprietary interest category: E, C₂.

 Presented in abstract form at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May, 1995.

Figure 1.

 

Clinical appearance of a typical intraocular 99E1 tumor treated topically with either anecortave acetate (A, C) or vehicle (B, D) on day 10 (A, B) or day 28 (C, D) after intracameral transplantation of 99E1 uveal melanoma cells.

Figure 2.

 

Clinical assessment (mean ± SEM) of the effect of topically applied anecortave acetate and vehicle on the intraocular growth of 99E1 tumors in BALB/c nude mice. Tumor growth rate in the anecortave acetate treated group was statistically slower than the growth rate in the control group (P < 0.025). n = 9 to 30 for the control group and n = 5 to 25 for the anecortave acetate treated group.

Figure 3.

 

Effect of topically applied anecortave acetate on intraocular tumor mass (mean ± SEM). There were 5 to 11 mice per group.* P < 0.05 for days 10, 21, and 28.

Figure 4.

 

Effect of anecortave acetate (Anecort Ac) and its deacetylated metabolite AL-4940 on the proliferation of 99E1 tumor cells. Addition of either compound at 0.1 μm or 1 μm for 24 to 72 hours had no effect on tumor cell proliferation (mean ± SD, n = 4).

 

The authors thank Mark Von Tress for his biostatistical analysis of our data.