Corneal penetration of topical and subconjunctival bevacizumab

Abstract

Purpose: To investigate the ability of bevacizumab to penetrate the cornea after topical application or subconjunctival injection.

Methods: Bevacizumab 1% was topically applied three times a day to the corneas of mice (BALB/c) with intact corneas (n = 14), and with corneal neovascularization (n = 14). Animals were euthanized at 1, 6, 12, and 24 hours, and 2, 4, and 7 days for immunohistochemical analyses. Donkey anti-human IgG labeled with Cy3 was used for bevacizumab immunoreactivity detection. Additionally, one-time topical bevacizumab 1% was tested in corneas with denuded epithelium (n = 16). In another group (n = 16), a single dose of 0.5 mg bevacizumab was injected subconjunctivally. Animals were euthanized at 1, 6, and 24 hours, and 2, 4, 7, 14, and 21 days for immunohistochemical studies.

Results: Bevacizumab was barely detected beyond the very superficial layer of the corneal epithelium in mice with intact corneas even after 7 days of topical administration. Application of bevacizumab in mice with corneal neovascularization; however, showed variable penetration into the corneal stroma. Experimentation with single application of topical bevacizumab in corneas with denuded epithelium or subconjunctivally injected bevacizumab showed intense staining for bevacizumab.

Conclusions: Topically applied bevacizumab has limited capacity to penetrate the corneas with intact epithelium. However, bevacizumab can penetrate the neovascularized cornea after topical application. This study demonstrates that subconjunctivally injected bevacizumab in eyes with an intact cornea penetrates well into the corneal stroma.

Figures

Figure 1.

Immunohistochemistry staining for bevacizumab in corneal stroma of control eyes. Donkey anti-human IgG labeled with Cy3 was used for bevacizumab immunoreactivity detection. (A) Strong immunoreactivity for bevacizumab in a cornea with intrastromal injection of bevacizumab used as a positive control. Arrow shows site of injection. (B) Section from an untreated eye was used as a negative control showing no immunoreactivity for bevacizumab (magnification, ×200).

Figure 2.

Penetration of topical bevacizumab in cornea with intact epithelium. (A) One day, (B) 4 days, and (C) 7 days after topical application of bevacizumab 1%, in frequency of 3 times a day. (D) Twenty-four hours of high frequency (every ½ hour for 8 hours; 16 times in total) application of topical bevacizumab 2.5%. Note that bevacizumab was detected merely on the very superficial layer of the epithelium (magnification, ×200).

Figure 3.

Penetration of topical bevacizumab in cornea with neovascularization. (A–C) Seven days after initiation of topical bevacizumab in corneas with neovascularization. Note that immunoreactivity for bevacizumab was noticeable in stroma; however, staining intensity varied considerably among the corneas (magnification, ×200).

Figure 4.

Penetration of topical bevacizumab in cornea with denuded epithelium. (A) Six hours, (B) 24 hours, (C) 2 days, and (D) 14 days after single application of topical bevacizumab 1% in corneas with denuded epithelium. Note strong staining of corneal stroma which was seen at all time points up to 14 days (magnification, ×100).

Figure 5.

Corneal penetration of bevacizumab after subconjunctival injection. (A) One hour, (B) 6 hours, (C) 24 hours, and (D) 14 days after subconjunctival injection of a single dose of 0.5 mg bevacizumab (0.02 mL of 25 mg/mL solution). Arrow: site of subconjunctival injection. Note that intracorneal diffusion of bevacizumab started into the corneal periphery adjacent to the injection site and by 24 hours of injection, strong bevacizumab staining was detected in the whole cornea which remained almost unchanged up to 14 days (magnification, ×20).

Figure 6.

Corneal penetration of bevacizumab after subconjunctival injection. (A) One day, (B) 7 days, (C) 14 days, and (D) 21 days after subconjunctival injection of a single dose of 0.5 mg bevacizumab. Note that strong bevacizumab staining remained almost unchanged in all layers of stroma over the next time points up to 14 days; however, on day 21 bevacizumab staining started fading from the corneal periphery (magnification, ×100).