Pharmacokinetics and retinal toxicity of various doses of intravitreal triamcinolone acetonide in rabbits

Drug preparations, animals, and animal experiments

The 4 mg/0.1 ml dose of TA was obtained directly from commercially available TA (40 mg/ml, Kunming Jida Pharmaceuticals, Kunming, China). The 8 mg/0.1 ml concentration of TA was obtained with the following method. Commercial TA of 0.2 ml was spun down in centrifuge for 5 min at 1698 ×g; thereafter, the half supernatant was removed, and the remnant was mixed. Methanol (high-performance liquid chromatography [HPLC] grade) was purchased from Merk (Hamburg, Germany).

Adult female and male New Zealand white rabbits (2.0–2.5 kg) were obtained from the Laboratory Animal Center of Tongji Medical College of Huazhong University of Science and Technology (Wuhan, China), and all experimental procedures adhered to the guidelines of the Chinese Animal Administration and the Association for Research in Vision and Ophthalmology Statement for the use of animals in ophthalmic and vision research.

All rabbits were anesthetized with an intramuscular injection of ketamine hydrochloride (30 mg/kg) and chlorpromazine hydrochloride (15 mg/kg). Topical anesthesia was achieved with tetracaine hydrochloride. Tropicamide was applied to dilate the pupils. A baseline eye examination including slit-lamp biomicroscopy, funduscopy with direct ophthalmoscope, and intraocular pressure (IOP) measurement were performed. Before intravitreal injection, paracentesis with removal of 0.1 ml of aqueous humor was performed to avoid an increase in IOP. Intravitreal injection of 0.1 ml solution was performed after a sterile eyelid speculum was placed, using a 30-gauge needle in the superior temporal quadrant about 2.5 mm posterior to the limbus.

Pharmacokinetics

Forty-two rabbits were randomly divided into two groups of 21 animals each. Group 1 was intravitreally injected with 4 mg/0.1 ml TA, and six eyeballs were obtained by euthanizing three rabbits with an intravenous injection of 100 mg/kg sodium pentobarbital at 0, 7, 14, 30, 45, 70, and 130 days after TA injection. Group 2 received intravitreal injection of 8 mg/0.1 ml TA, and six eyeballs were obtained at 0, 10, 20, 35, 60, 90, and 150 days after TA injection.

Each eyeball was enucleated and immediately frozen in a −60 °C environment. The vitreous was isolated using the previously described technique by separating frozen vitreous from the sclera, retina, lens, cornea, and so on [13]. Each eyeball was enucleated and immediately frozen in a -60 °C environment, and the frozen vitreous was harvested carefully by separating sclera, retina, lens, cornea, and so on [13]. Vitreous samples were subjected to ultrasonic processor for 20 s at 0 °C (mixture with water and ice). The mixed vitreous of 0.1 ml was pretreated with the addition of 4.9 ml methanol. The mixture was blended and centrifuged at 1698 ×g for 30 min, and 20 μl of each supernatant was directly injected into reversed-phase HPLC (Shimadzu LC-10AD liquid chromatography equipped with SPD-10A UV detector, class-10A workstation system, Rheodyne injector, and 20-μl sample loop) to analyze the data. The Diamonsil C-18 chromatographic column (5 µm particle size, 250 mm × 4.6 mm, Dikma Technologies, Beijing, China) was used for separation, and detection was set at 254 nm. The mobile phase was composed of methanol:water (70:30) at a flow rate of 1.0 ml/min. The column oven was set at 40 °C. The TA retention time was 6.8 min, and the detection limit was 0.005 mg/ml. The standard curve was linear from 0.005 to 0.100 mg/ml (correlative >0.9998), using seven different amounts of TA standard. The concentrations of 0.01 mg/ml, 0.06 mg/ml, and 0.10 mg/ml TA in vitreous were measured for the recovery and intra- and inter-day reproducibility.

Retinal toxicity

Twenty rabbits were divided into two experimental groups: Ten right eyes in group A were injected with 4 mg/0.1 ml TA, and ten right eyes in group B were injected with 8 mg/0.1 ml TA. All left eyes were injected with 0.1 ml physiologic salt solution (PSS) as the control group.

IOP measurement was performed with a Schiötz ophthalmotonometer (Suzhou, China) before intravitreal injection and at 1, 4, 8, and 12 weeks following injection. All animals were kept in a quiet environment for about 30 min and then received topical anesthesia. After the eyelid was separated, the central part of the cornea was gently touched with the Schiötz ophthalmotonometer.

Electroretinography (ERG) was performed before intravitreal injection and at 1 and 3 months after injection using the method previously reported [27]. Briefly, the rabbits received dark adaption for at least 30 min before anesthesia and pupillary dilatation with the described methods. A silver electrode was placed on the cornea and served as a positive electrode. The reference electrode was placed subcutaneously between the base of the ear and the lateral canthus. The ground electrode was placed subcutaneously at the forehead area. The ERG included 30 Hz flicker, scotopic 0.5 Hz every 5 min during the 30 min of dark adaptation, with a flash intensity of 3.0 cd•s/m².

The animals were euthanized, and both eyes were enucleated at 3 months after the last ERG. Enucleated eyes were fixed in 10% formalin for light microscopic examination, or 2.5% glutaraldehyde for transmission electron microscopy. Light microscopic sections were stained with hematoxylin-eosin, and transmission electron microscopic sections were stained with lead and uranyl acetate.

Statistical analysis

The elimination half-life (t_1/2), initiate theoretical concentration (Co), and area under the concentration time curve (AUC) were calculated with 3p97 pharmacokinetics software (Practical Pharmacokinetics Software, Beijing, China). Using SPSS 13.0 (SPSS Inc., Chicago, IL), the IOP and ERG b-wave amplitude data were compared by using repeated measures ANOVA. p<0.05 was considered significant.

Pharmacokinetics

Chromatograms of TA in samples are shown in Figure 1. TA was separated clearly and was not interfered by the other substances. The recovery and intra- and inter-day reproducibility results are shown in Table 1. The mean recovery from the vitreous was 98.36%. These data suggested that TA could be efficiently extracted from the vitreous via this method, and analyzing the TA concentration of intravitreal injection was reliable.

Figure 2 shows the time course of the TA concentration in the vitreous of the rabbits following intravitreal injection of 4 mg and 8 mg TA. The concentrations of both doses decreased gradually with time, and the peak concentration of intravitreal TA was the initial concentration. A concentration of 0.0524 mg/ml was maintained in the vitreous at 130 days after the injection of 4 mg TA, and 0.2606 mg/ml was detected at 150 days following the intravitreal injection of 8 mg TA. The calculated half-lives of intravitreal TA were 24 days for the 4 mg dose and 34 days for the 8 mg dose. The initiate theoretical concentration of intravitreal 4 mg and 8 mg TA were 2.745 mg/ml and 5.498 mg/ml, and the AUC were 95.560 (mg/ml)/d and 247.789 (mg/ml)/d, respectively. These data show that intravitreal injection of 8 mg TA achieved a longer half-life and higher concentration and bioavailability compared with the intravitreal injection of 4 mg TA.

Retinal toxicity

The vitreous was transparent with the intravitreal injection of PSS, and TA was visible as a white mass in the vitreous after the injection (Figure 3). The size of the mass became gradually smaller. No endophthalmitis, keratic damage, cataract, vitreous hemorrhage, or retinal detachment was discovered during the study period. The IOP and b-wave amplitudes for each group at different time points are shown in Figure 4 and Figure 5. There was no significant difference in IOP between any groups (F = 0.316, p = 0.732>0.05) or between any time points (F = 0.835, p =0.507>0.05), and there were no significant differences between any groups (F = 1.868, p = 0.167>0.05) or between any time points (F = 0.079, p = 0.925>0.05) in the ERG b-wave amplitudes. The histopathologic examination of both groups showed no structural abnormalities under light microscopy at 3 months, and normal layers of retina were revealed in both groups (Figure 6). Transmission electron microscopy confirmed these findings and demonstrated a complete normal retina in each group. The outer nuclear layer and the inner nuclear layer are normal in Figure 7A,B. The inner and outer segments of the photoreceptors are also normal at 3 months in Figure 7C,D. These results suggest that intravitreal injection of 4 mg and 8 mg TA is safe for the rabbit retina.