Evaluation of 9-cis-retinyl acetate therapy in Rpe65-/- mice

Abstract

Purpose: Mice lacking retinal pigment epithelium-specific 65-kDa protein (RPE65) develop retinopathy and blindness resembling Leber congenital amaurosis. Effects of 9-cis-retinyl acetate (9-cis-R-Ac) on visual function and retinopathy progression were tested in Rpe65(-/-) mice.

Methods: Young C57Bl/6 mice were given 9-cis-R-Ac in each of four different oil-based vehicle solutions by gastric gavage to identify the vehicle most suitable for drug delivery by measuring retinoid levels in plasma. Then doses of 9-cis-R-Ac ranging from 1 to 100 mg/kg were administered to 5- to 12-week-old Rpe65(-/-) mice by different treatment regimens, including single doses and either intermittent or daily doses for various periods up to 8 weeks. Retinoid effects on visual function were evaluated by electroretinography, retinoid analyses, histologic methods, and vision-dependent behavioral testing.

Results: Soybean oil vehicle provided the highest 9-cis-R-Ac metabolite levels in plasma. Single doses of 9-cis-R-Ac (6.25-50 mg/kg) provided significant dose-dependent improvement in electroretinographic responses. Well-tolerated daily doses (1-12.5 mg/kg) for 2 weeks induced remarkable improvement of retinal function. Significant dose-dependent improvement of electroretinographic responses was observed 6 days after administration of 9-cis-R-Ac daily for 3 days at 1 to 12.5 mg/kg. Mice given either daily or intermittent 9-cis-R-Ac treatment at 1 and 4 mg/kg and evaluated 8 weeks later displayed dose-dependent improvement of retinal function and morphology, whereas retinal function deteriorated in control animals. Treated mice also performed better than control animals in vision-dependent behavioral tests.

Conclusions: Treatment with 9-cis-R-Ac improves visual function and preserves retinal morphology in Rpe65(-/-) mice.

Figure 1

Solubilizing vehicle effects on plasma retinoid levels in mice gavaged with 9-cis-R-Ac. A single 50-mg/kg dose of 9-cis-retinyl acetate (9-cis-R-Ac) suspended in four different vehicle oils was administrated by gastric gavage to 5-week-old C57/Bl6 mice, and retinoid levels were determined in the plasma thereafter (n = 5 for each time point per group). (A) Fatty acid 9-cis-retinyl esters. (B) Fatty acid all-trans-retinyl esters. (C) 9-cis-retinol. (D) All-trans-retinol levels were determined at five sequential time points. 9-cis-Retinoid levels in plasma were significantly higher and were maintained for 3 hours after administration, when 9-cis-R-Ac was given in either soy-bean or sunflower oil. Bars indicate SD.

Figure 2

Improvement of retinal function in Rpe65^−/− mice given single doses of 9-cis-R-Ac. Five-week-old Rpe65^−/− mice underwent gavage with single doses of 9-cis-R-Ac ranging from 2 to 50 mg/kg and then were maintained for 3 days in the dark (n = 2 at 50 mg/kg; n = 3–4 for other groups). Scotopic single-flash ERGs were recorded from these mice (A, B; bars indicate SE), and 9-cis-retinal levels were quantified in their eyes (C; bars indicate SD). ERGs showed a dose-dependent positive response for a- and b-wave amplitudes in mice given 9-cis-R-Ac, and 9-cis-retinal accumulated in their eyes in a similar manner. * Undetectable amounts.

Figure 3

Experimental protocol for 14-day daily treatment of Rpe65^−/− mice with 9-cis-R-Ac. Groups of 5-week-old Rpe65^−/− mice underwent gavage daily for 14 days with different doses of 9-cis-R-Ac (1, 4, or 12.5 mg/kg; n = 4–6 per group). Mice were kept in the dark from day 1 to day 3 and then were maintained in an 8-hour light/16-hour dark cycle environment from day 4 to day 14, when ERGs were recorded and eyes were analyzed for retinoids.

Figure 4

Improvement of retinal function in 5-week-old Rpe65^−/− mice after gavage with daily doses of 9-cis-R-Ac for 14 days. Scotopic single-flash ERGs from these mice were obtained on day 14, and functional a-and b-wave amplitude plots are shown (A, B). ERG responses improved in a dose-dependent manner. Bars indicate SE (n = 4–6 per group except baseline; baseline, n = 2). Eye levels of 9-cis-retinoids also are shown for day 14 (C, D). 9-cis-Retinoids accumulated in a dose-dependent fashion in the eyes of mice treated with 4 and 12.5 mg/kg 9-cis-R-Ac, but these compounds were not detectable (*) in mice treated with the lowest dose (1 mg/kg) or left untreated (baseline). Bars indicate SD (n = 4–6 per group except baseline; baseline, n = 2). ERG wave amplitudes and retinoid levels in the eyes of treated mice were compared with similar data obtained from base-line mice. The key (A) indicates doses of 9-cis-R-Ac, as do numbers under the bars (C, D).

Figure 5

Experimental protocol for assessment of retinal function after daily administration of 9-cis-R-Ac for 3 days. This experiment was designed to test the duration of visual improvement after the completion of brief treatment with 9-cis-R-Ac. Groups of 5-week-old Rpe65^−/− mice were given 9-cis-R-Ac by oral gavage (12.5, 4, or 1 mg/kg) or soybean oil for 3 days (days 1–3) and were exposed daily to fluorescent light with a luminance range of a 500 to 1500 lux in the cage for 8 hours before they were returned to the dark. Retinal function was evaluated by ERGs, and eye retinoid level analyses were conducted on the first (day 4), second (day 5), fourth (day 7), and sixth (day 9) days after the completion of 9-cis-R-Ac therapy, as shown.

Figure 6

Duration of improvement in retinal function after 3-day administration of 9-cis-R-Ac. When 8-hour fluorescent light exposure ended at 4 pm, single-flash scotopic ERGs were recorded at four sequential time points to evaluate the duration of improvement in retinal function after treatment with 9-cis-R-Ac (Fig. 5; n = 3–4 per group except for day 9, at a dose of 12.5 mg/kg, and control day 9, at n = 2 each). Bars indicate SE. ERG responses of 9-cis-R-Ac treated and control mice were plotted, and functional a- and b-wave amplitudes are shown: (A, B), 12.5 mg/kg; (C, D), 4 mg/kg; (E, F), 1 mg/kg. Even the lowest dose (1 mg/kg) significantly improved b-wave amplitudes of ERG responses at high-intensity stimuli on day 9 compared with responses of control mice. P < 0.05.

Figure 7

Experimental protocol for examining the effects on retinal function of intermittent and daily administration of 9-cis-R-Ac to 5-week-old Rpe65^−/− mice for 8 weeks. Groups of Rpe65^−/− mice were treated for 8 weeks with either 1- or 4-mg/kg daily doses of 9-cis-R-Ac for 3 days followed by 4-day weekly drug holiday periods (intermittent group, n = 6–7 per group), or the same two doses were given daily (daily group, n = 6–7 per group). Mice were maintained under an 8-hour fluorescent light (luminance range, 500–1500 lux in the cage)/16-hour dark cycle during the 8-week experiment. ERGs were recorded on days 28 and 56, and the tissues were removed for histology and retinoid analyses of eyes and liver on day 56.

Figure 8

Effects of intermittent and daily administration of 9-cis-R-Ac on ERG responses of Rpe65^−/− mice recorded on days 28 and 56. Dose-dependent improvement in the amplitude of a- and b-waves is shown with both treatment regimens at day 28: (A, B), intermittent treatment; (C, D), daily treatment; n = 5–7 per group; and day 56: (E, F), intermittent treatment; (G, H), daily treatment; n = 5–7 per group. ERG responses of both groups of treated mice were superior to those from controls. Bars indicate SE.

Figure 9

Retinal morphology of 9-cis-R-Ac–treated mice. ROS lengths and numbers of nuclei in the outer nuclear layer (ONL) of treated and control mice are shown at day 56. Data points are plotted as a function of the distance from the optic nerve head (ONH). Lengths of the ROS (A, intermittent treatment; C, daily treatment) and the number of nuclei in the ONL (B, intermittent treatment; D, daily treatment) in the superior retina were significantly greater in a dose-dependent manner for 9-cis-R-Ac–treated mice compared with control groups. These positive effects were more prominent in mice treated at the 4-mg/kg dose, whereas positive effects were observed only in limited areas in mice treated with the 1-mg/kg dose (n = 3–4 per group). Bars indicate SD. Images of the ROS and RPE interface in control and treated mice at day 56 are shown: (E), 4 mg/kg intermittent treatment; (F), 4 mg/kg daily treatment; (G), 1 mg/kg intermittent treatment; (H), 1 mg/kg daily treatment; (I), control. Improved ROS structures and fewer oil droplet-like structures were present in mice treated with 4 mg/kg 9-cis-R-Ac compared with control mice Arrows: oil droplet-like structures in (I). Scale bar, 5 µm.

Figure 10

Improvement of vision-dependent behavior in Rpe65^−/− mice treated with 9-cis-R-Ac. Twelve-week-old Rpe65^−/− mice were subjected to three different vision-dependent behavioral tests—a dark/light preference test, a visual placement test, and a water escape task—after they had received 9-cis-R-Ac either as a single 100-mg/kg dose or as 12.5 mg/kg daily for 3 days (n = 3 per group). In the dark/light preference test, treated mice moved to a dark space from an illuminated area significantly faster than control mice (A). The visual placement test also demonstrated a significantly higher success rate of reflexive arm extension in response to visualization of a counter edge in treated compared with control mice (B). In the water escape task, treated mice took significantly less time (average 32% less at 100 mg/kg, 25% less at 12.5 mg/kg) to find the hidden platform by using a light as a cue in a dose-dependent manner, whereas improvement in escape latencies was not observed in control mice (C). Bars indicate SD.