Evaluation of Hydrogel Bandage and Eye-Contact Devices for HSV-1-Glycoprotein-D Delivery Against Keratitis Formation in

Rabbit Ocular HerpesChristian Clement, PhD, MPhil1,a, AuthorVaibhav Tiwari, PhD2, Co-AuthorHilary W. Thompson, PhD2 1,3-6, Co-AuthorDepartments of 1Ophthalmology, 3Pharmacology, 4Microbiology, 5Neuroscience Center, 6School of Public Health Biostatistics, Center of Excellence, Louisiana State University Health Sciences Center School of Medicine, New Orleans, LA; 2College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, Pomona, CAFinancial Disclosurea Ocular Therapeutix, Inc. Travel SupportResure™ Adherent Ocular Bandage (novel hydrogel bandage) is a courtesy of Ocular Therapeutix, Inc., Bedford, MA.Potential corneal toxicity to ReSure™-not assessed as it is CE Mark approved for use in Europe and is investigational in the U.S.This study was presented in part at the AAO-MEACO October 16-19, 2010 Chicago, IL

PURPOSENovel hydrogel bandage and eye-contact devices for drug delivery and as physical protective barrier

1. To evaluate the course of corneal lesions and keratitis formation in eyes of rabbits infected with herpes simplex virus type-1 (HSV-1) and treated with an in-situ forming hydrogel bandage, contact-lens and collagen-soft-shield each containing HSV-1 glycoprotein D (gD) added to block virus infection sites as antiviral-replication strategy

2. Reduction of Pseudomonas aeruginosa keratitis in a rabbit keratotomy model by novel hydrogel bandage (AAO 2010-PO041) has been summarized.

PURPOSE continuedStrategy of delivering crucial viral protein to eye surface to prevent infection HSV-1 virus has a glycoprotein, gD which it uses to physically bind to co-receptors on the surface of cells/tissues in infection. We expressed the non viral protein (61KDa) and added this ligand at ~10 nanograms/µl to hydrogel bandage and eye-contact devices for delivery to corneal surface to prevent actual infectious virus from binding and infecting.

Confocal microscopy showing actual HSV-1 gD (red) physically bound to cell surface receptor (green) during viral infection process. Courtesy V. Tiwari et al FEBS letters 581 (2007) 4468-4472.

Constituents of Novel Hydrogel Bandage

The novel hydrogel bandage, supplied in separate syringes is reconstituted as liquid preceding gelation. The novel hydrogel bandage is composed of 3 components all of which have a long history of safe ophthalmic use- polyethylene glycol, trilysine, and a pH-balanced aqueous accelerator solution.

Novel hydrogel bandage use- Six simple steps

4. Accelerator added to drop of solution

5. Two drops mixed with single-use applicator

6. Droplets applied to ocular surface within less than 25 seconds.

1. Two syringes are attached

2. Powder + diluent mixed

3. Drop of solution colored blue (for visual aid) dispensed

MATERIALS AND METHODS(Herpetic keratitis)

• A prospective nonrandomized comparative study.• 16 NZW rabbits of the same age and in 4 groups (hydrogel bandage,

Acuvue® contact-lens, Oasis® soft-collagen-shield and Untreated).• Corneas were each scarified and separate inoculations done with

2x105 PFU of infectious HSV-1, 17Syn+.• Initial slit-lamp examination (SLE) was at post infection (PI) day 2

followed by applying on eyes HSV-1-gD impregnated, bandage, other contact devices secured by partial tarsorrhaphy , and untreated.

• All eyes were scored by SLE for appearance of corneal lesions and corneal opacity from PI day 3 to PI day 28 (graded scale: 0, no opacity; 1, mild cloudiness with visible iris; 2, moderate cloudiness with obscured iris; 3, total corneal cloudiness with invisible iris and 4, total opacity with no posterior view).

MATERIALS AND METHODS(Pseudomonas Keratitis)

• A prospective nonrandomized comparative study.• 16 NZW rabbits of the same age and in 4 groups.• Keratotomy was performed on 1 eye of all rabbits by making cuts

to the right central corneas (cross incisions, 5x5mm; depth, 0.2mm).

• Control (8 eyes), P. aeruginosa-soaked collagen shield was placed over the incision.

• Experimental (8 eyes), novel hydrogel bandage was applied before P. aeruginosa-soaked collagen shield placed over the incision.

• All collagen shields were secured by partial tarsorrhaphy and remained in place for 20 or 44 hours.

• CFU were determined by plating 10-fold serial dilutions of homogenized corneas surgically removed from euthanized rabbits.

RESULTS(Herpetic keratitis)

HSV-1 gD delivery by novel hydrogel bandage and eye-contact devices

Hydrogel bandage-gD treated eye,average score 0.5, no detectable keratitis

Lens-gD treated eye, average score 2.0, mild keratitis

RESULTS continued (Herpetic keratitis)

Shield-gD treated eye, average score 3.5, with keratitis and corneal neovascularization

Untreated eye, average score 4.0, with severe keratitis and corneal neovascularization

RESULTS continued(Pseudomonas keratitis)

Novel hydrogel bandage treated versus untreated eyes

Infected-treated eye (44 hours), substantial protection against inflammatory and bacterial infiltration in cut area

Infected-untreated eye (20 hours), engulfment of cornea by discharge and exudates

Infected-untreated eye (44 hours), inflammation, injection (arrow) and friable neovascularization (arrowhead)

Colony forming units/mL in novel hydrogel bandage treated versus untreated eyes

Test condition Mean CFU/mL

% change p value 20 h 44 h

Control 6.6 ± 0.47 x 106 7.85 ± 3.64 x 106 +19% 0.1730

Hydrogel bandage 3.7 ± 1.24 x 106 0.86 ± 0.24 x 106 -77% 0.0336

Mean CFU/mL from treated and untreated eyes after 20 or 44 hours incubation with bacteria-soaked collagen shields.

Comparison of CFU/mL, treated and untreated eyes across post-infection time points.

DISCUSSION AND CONCLUSION

• Incisions to the eye are conduit for infection.• In general use of natural molecules (proteins) to bind and block sites on and within

cells/tissues that facilitate pathogen entry are complicated.

• Novel hydrogel bandage treated corneas exhibited significant protection against pathogen entry.

• Novel hydrogel bandage and contact lens delivered viral protein gD which protected

incisions from acting as sites for pathogen exploitation for establishment of infection.

• Novel hydrogel bandage applied over wounds in rabbit corneas, also protected against P. aeruginosa infection.

• Novel hydrogel bandage and contact lens could successfully be modified for healing surgical wounds in grafting and penetrating keratoplasties and could be ideal for lowering the risk of bacterial infection in cataract surgeries and vitrectomies.