Development of a biodegradable antifibrotic local drug delivery system for glaucoma microstents

Abstract

To prevent implant failure due to fibrosis is a major objective in glaucoma research. The present study investigated the antifibrotic effects of paclitaxel (PTX), caffeic acid phenethyl ester (CAPE), and pirfenidone (PFD) coated microstent test specimens in a rat model. Test specimens based on a biodegradable blend of poly(4-hydroxybutyrate) biopolymer and atactic poly(3-hydroxybutyrate) (at.P(3HB)) were manufactured, equipped with local drug delivery (LDD) coatings, and implanted in the subcutaneous white fat depot. Postoperatively, test specimens were explanted and analyzed for residual drug content. Fat depots including the test specimens were histologically analyzed. In vitro drug release studies revealed an initial burst for LDD devices. In vivo, slow drug release of PTX was found, whereas it already completed 1 week postoperatively for CAPE and PFD LDD devices. Histological examinations revealed a massive cell infiltration in the periphery of the test specimens. Compact fibrotic capsules around the LDD devices were detectable at 4-36 weeks and least pronounced around PFD-coated specimens. Capsules stained positive for extracellular matrix (ECM) components. The presented model offers possibilities to investigate release kinetics and the antifibrotic potential of drugs in vivo as well as the identification of more effective agents for a novel generation of drug-eluting glaucoma microstents.

Keywords: caffeic acid phenethyl ester; fibrosis; glaucoma; microstent; paclitaxel; pirfenidone.

Figure 1. Illustration of the microstent concept and photo documentation of the minimally invasive surgical intervention

(A) Schematic drawing of a microstent for the drainage of aqueous humor from the anterior chamber of the eye into the retro-orbital intraconal fatty tissue; microstent with micro-mechanical valve for the prevention of hypotony (*) located in the anterior chamber and LDD coating (**) for the prevention of fibrosis located in the outflow area. (B) Implantation of test specimen into the subcutaneous white fat depot of rats and explantation procedure. (a) Shaved and disinfected implantation area with mark for incision. (b) Carefully opened cutis. (c) Test specimen injection into white fat depot using a PICO-ID-Chip-Injector. (d) Wound closure by suture. (e) With two stitches closed cutis. (f) Explantation of test specimen. Arrow marks the implant.

Figure 2. Manufactured test specimens and quantitation of drug release in vitro and in vivo

(A) Representative scanning electron micrographs of test specimens from (a) group 1 (no LDD coating), (b) group 2 (PTX LDD coating), (c) group 3 (CAPE LDD coating), and (d) group 4 (PFD LDD coating) at 150× and 400× magnification, respectively. (B) Cumulative drug release from different LDD devices in vitro in an initial time frame of the release studies (each n=4, normalized to recovered drug mass). (C) Drug release from different LDD devices in vitro and in vivo within 12 weeks (each n=4).

Figure 3. Histology of the subcutaneous white fat depots including the test specimens

(A) Cross-sections of the rat white fat depots were stained for connective tissue using AZAN staining. A connective tissue-rich fibrotic capsule is obvious after 6 months in the periphery of uncoated control implants, as well as in the PTX-, CAPE-, and PFD-coated specimens. (B) Cross-sections were H&E stained. Comparable with (A), strongest implant encapsulation is obvious after 6 months. Bars represent 200 µm.

Figure 4. Evaluation of the fibrotic tissue response in comparison with drug release data in vivo

Histological sections (Figure 3A) were used to measure the thickness of fibrotic capsules (left y-axis) surrounding uncoated and drug-coated test specimens. Results are depicted as colored bars (n=3; mean ± S.D.). Drug release data of coated test specimens (right y-axis), as determined with HPLC, are depicted as curves in corresponding colors (n=4, mean). After 1 week no residual drug could be quantitated for CAPE- and PFD-coated test specimens. Fibrotic tissue reactions for these two coatings were visible after 4 weeks, whereas PTX-coated test specimens showed fibrotic capsules after 12 weeks. In uncoated samples, fibrotic capsules were observed starting 2 weeks after implantation. Mean ± S.D. (each n=3).

Figure 5. Immunohistochemical evaluation of the fibrotic tissue response to test specimens

(A) The cross-sections of the rat fat depots including the test specimens were stained for collagen I. The connective tissue-rich fibrotic capsule is positively stained for collagen I with lowest expression in the PFD group. (B) Immunohistochemical examinations of cross-sections stained for collagen VI revealed high reactivity of the connective tissue-rich fibrotic capsules, which ensheath the implants with lowest reactivity in the PFD group. Bars represent 200 µm.

Figure 6:. Immunohistochemistry of the subcutaneous white fat depots including the test specimen implants

(A) Immunohistochemical reactivity against fibronectin could be detected after 6 months around the uncoated control and the drug-coated implants with weakest signal in the PFD group. (B) The cross-sections of the rat fat depots including the test specimens were stained for CD11b. Expression could be detected around the uncoated control and the drug-coated implants. Bars represent 200 µm.