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Data statistics and calculations were performed on Microsoft Office Excel. Table 1. View Table. The ophthalmic irritation signs were evaluated at predosing, 1 hour after each dose and 24 hours after the initial dose by clinical observation and slit-lamp microscopy. To mimic pigmented human eyes in the event that the drug binds to melanin, the pigmented DB rabbits were utilized for nAMD model establishment and evaluation of therapeutic efficacy. Pigmented rabbits were sedated with 0.

The animal eyes were topically anesthetized with 1 to 2 drops of 0. Antibiotic ointment was administered to the lower lid of the eyes. Pharmacodynamic Evaluation of the Obtained Eye Drop. Avastin group received one dose of intravitreous bevacizumab Avastin. After the anesthetized procedure as previous described, the fluorescein was administered into the marginal ear vein. Photographs of the fundus were taken within 10 minutes after the fluorescein injection. Leakage area was quantitatively measured by fluorescein angiography image analysis based on brightness using open-source software ImageJ to assessment the pharmacodynamics.

Then, all animals were adminstered 2. Eylea group received one dose of intravitreous aflibercept. Fundus Photography and Fluorescein Angiography. The cornea was anesthetized with topical Alcaine 0.

Methylcellulose eye gel was applied to prevent the dehydration of the cornea during imaging. Following a subcutaneous injection of 0. Images were exported as tagged image files. Optical Coherence Tomography Acquisition and Analysis. After general anesthesia, the rats were placed on the imaging platform, and the head was positioned at an angle to allow the penetration of light vertical to the cornea from the temporal side.

The retinal structure was obtained by linear scanning around the lesion site. At least three clear captures were obtained for each eye. The center of the lesion was defined as the midline passing through the area of RPE-Bruch's membrane rupture. OCT was only utilized for monitoring the CNV model establishment, especially for the bubble formation in Bruch's membrane. The rat eye with the lesions containing no bubble or bubble with hemorrhage was excluded from this study. For single pairwise comparisons, Student's t -test was applied.

Though the aqueous solubility of axitinib is rather low, approximately 0. Similar phenomena have also been observed in the other eye drop formulation as shown in our previous work.

The pH, osmolality, and viscosity of the eye drop formulation were measured to be 6. Detailed stability data was described in Table 2. Table 2. The exposure criteria was set fold above the half maximal inhibitory concentration IC 50 of axitinib fold of axitinib IC 50 : 7.

These results demonstrate efficient drug delivery to posterior ocular tissues of our formulation. Figure 1. View Original Download Slide.

Cynomolgus monkeys were also utilized for the ocular pharmacokinetic study because only primates have a macula. Following topical ocular administration of ITRI AXN eye drops to the monkey eyes three times daily, the mean concentrations of axitinib in aqueous humor, choroid-RPE, cornea, iris-ciliary body, lens, retina, sclera, and vitreous humor at each time interval were shown in Table 3.

The accuracy of quality control samples was between The results showed that the drug level in the retina and choroid remains high and above preset therapeutic concentrations over the time period of 24 hours, except for the 24 hour time point of retina, which was 5.

Drug retention in the choroid and iris-ciliary body was observed, which is likely attributed to the melanin binding of axitinib in pigmented monkeys. Table 3. The safety of ITRI AXN eye drops in terms of the acute ocular irritation and corrosion was evaluated on the rabbits which received the eye drop three times daily.

The assessment was performed in accordance with the test guideline of OECD As shown in Figure 2 , all rabbits receiving ITRI AXN eye drops at preset time intervals demonstrated a rather benign effect on rabbit eyes without any ocular irritation and corrosion, as evidenced by a lack of apparent side effects on corneal structure and integrity average total score 0.

These data strongly suggest the nonirritant property of the obtained formulation. Figure 2. Representative pictures of the in vivo rabbit ocular irritation test. Antiangiogenesis Effect on Animal Disease Model. VEGF protein was given intravitreally at day 0 to induce retinal vascular leakage to simulate exudative neovascular agerelated macular degeneration nAMD.

The fluorescein, a fluorescent tracer, was intravenously administered for observation of blood leakage by fundus fluorescence angiography. As shown in Figure 3 a, significant blood leakage at either optic disc or medullary wings was observed 2 days post VEGF injection, suggesting the successful production of a disease model.

This phenomenon persisted at least for 2 days from day 2 to day 4. The substantially reduced intraocular VEGF level is inadequate to generate angiogenesis and alter vascular permeability in retina.

The results obviously demonstrated the superior therapeutic potential of the eye drop approach for nAMD treatment. Figure 3. The laser-induced CNV model, the most commonly used and mainstay model for nAMD therapeutic evaluation, was utilized to evaluate the dose effects.

Six laser spots were applied in the rat's eyes. Laser injury on Bruch's membrane located between the retinal pigment epithelium and the capillary lamina of the choroid induces angiogenesis capable of modeling the hallmark pathology of neovascular AMD. The angiogenesis progression in each group was monitored at day 3 and day 14 post laser-induced CNV induction. Data revealed that six CNV lesions could be observed in each group without significant hemorrhage at day 3 Fig.

A great reduction of blood leakage was observed at day 14 in the group intravitreally injected with Eylea, while the fluorescein signal of the group topically receiving the vehicle remained high. This indicates the pronounced antiangiogenic effect of anti-VEGF antibodies. Moreover, the higher the drug level in the eye drop formulation, the better the antiangiogenic effect until therapeutic plateau was reached for the 0.

There is no statistically significant difference between the groups of Eylea and 0. These results clearly demonstrated the great potential of the eye drop approach for replacement of anti-VEGF injections in nAMD treatment.

Figure 4. VEGF is the key mediator of angiogenesis in nAMD, which leads to abnormal blood vessels that leak fluid or blood into the macula. In this study, we formulated an eye drop with VEGF inhibitor, termed ITRI AXN eye drops, and the pharmacokinetic and pharmacodynamic results showed that sufficient drug reached the choroid and retina leading to similar treatment efficacy as intravitreal injected VEGF antibody.

This may be clinical translational relevance in ophthalmology and medicine. The drug aqueous solubility was substantially enhanced from ca 0. The pharmacokinetic studies of rabbits and NHP showed that the drug exposure at the retina and choroid was to fold greater than IC 50 of drug after topical administration of the eye drops. It should be noted that the other eye drop formulations have also shown promise in ocular drug delivery in preclinical studies but failure in clinical trials due to lack of efficacy.

Horita et al. Joussen et al. To this end, the drug exposure threshold of fold drug IC 50 axitinib IC 50 : 7. Consistent with the pharmacokinetic results shown above, the effective antiangiogenic effect on target tissues by using ITRI AXN eye drops were demonstrated in rabbits and monkeys.

To evaluate therapeutic potential of the eye drop approach, the preclinical animal models of retinal and choroidal neovascularization, which have been considered as major features of nAMD, were employed in this work. It should be noted that melanin is an important pigment widely existing in human ocular tissues, such as the iris, ciliary body, choroid, and retinal pigment epithelium.

VEGF was intravitreally injected into the rabbit eyes for induction of the retinal neovascularization model. After being treated with the eye drop formulation, a comparable antiangiogenic outcome to the group injected intraocularly with anti-VEGF antibody to neutralize VEGF was observed.

This demonstrates the great antiangiogenic potential of the eye drop approach for nAMD treatment. Antiangiogenesis study on the other nAMD model of choroid neovascularization, which is the mainstay model for nAMD therapeutic evaluation, was also performed. Again, the eye drop formulations showed a significant antiangiogenic effect, even in those low-dose groups 0. The decrease of blood vessel leakage in the choroid was associated with elevated drug levels in eye drop formulations.

Maximum therapeutic effect was observed in the group of 0. Ocular irritation assessment was also evaluated on rabbits. After receiving the eye drop formulation three times daily, the score for ocular lesions is zero, reflecting that the rabbit eyes remained rather benign without occurrence of acute irritation and corrosion. In conclusion, ITRI AXN eye drops demonstrated a great potential in drug delivery to posterior ocular cavity with a rather high-level drug exposure at the retina and choroid.

By virtue of the sufficient therapeutic level in target sites of nAMD, the obtained eye drop formulation shows a great promise in antiangiogenic outcome against the retinal and choroidal neovascularization model in vivo while eye irritation remains rather benign. These results suggested that the goal of this study can be successfully achieved by our formulation. We believe further clinical studies are justified. Disclosure: W. Huang, None; F. Cheng, None; C. Chen, None; P.

Kuo, None; Y. Wang, None; S. Yin, None; C. Tu, None; M. Wu, None; W. Chen, None. Effects of aflibercept for neovascular age-related macular degeneration: a systematic review and meta-analysis of observational comparative studies. Stop use and ask a doctor if you experience any of the following:. Keep out of reach of children. If swallowed, get medical help or contact a Poison Control Center right away. Buy Now. Provides fast hydration and tear evaporation protection for all major types.

Get personalized dry eye product recommendations and helpful tips to reduce symptoms and triggers that may impact your eyes. Buy Now Get Coupon. Product Details. Drug Facts. Active Ingredients Purpose Polyethylene Glycol 0. For external use only. Stop use and ask a doctor if you experience any of the following: eye pain changes in vision continued redness or irritation of the eye condition worsens or persists longer than 72 hours Keep out of reach of children.

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