780-VAD-FMK Tracer With No Light Damage Focus on Retinal Vasculature Focus on RPE/Choroid OCT 2 Days Post Blue Light Damage 780-VAD-FMK Tracer With Light Damage Focus on Retinal Vasculature Focus on RPE/Choroid OCT 10 Days Post Blue Light Damage

CAS-MAP P 780 in vivo, (780-Val-Ala-Asp(OMe)-Fluoromethyl ketone, or 780-VAD-FMK) is a cell permeant tracer designed to detect and image apoptosis in vivo by binding to active caspases. The reagent is injected intravenously and allowed to circulate. The low molecular weight (976.2) and chemical characteristics allow it to transverse the cell membrane and the blood retinal barrier. If cells have active caspases, the main mediator of apoptosis, they are in a state of apoptosis. Once inside the cell the CAS-MAP P 780 in vivo reagent will form a covalent bond to the sulfhydryl group of a cysteine residue in the active catalytic site of the caspase protease. The reagent remains in the cell once it is bound to an active caspase. Unbound CAS-MAP P 780 in vivo reagent, in healthy cells, is quickly washed out and eliminated by normal physiological processes. The remaining caspase-bound reagent can then be imaged by exciting at 775 nm and reading at 802 nm. This results in a sensitive and specific method of apoptosis detection and imaging apoptosis in the retina.

780-VAD(OMe)-FMK Spectrum

CAS-MAP P 780 in vivo 780-Val-Ala-Asp(OMe)-Fluoromethyl ketone

Binding Mechanism of 780-VAD-FMK to the Sulfhydryl group of a Cysteine residue in the Catalytic site of Active Caspases

Formation of thiohemiketal (Intermediate I): Formation of a 3 membered sulfonium ring (Intermediate II) Final rearrangement to give a thioether adduct and a stable covalent bond between the 780-VAD-FMK and the active caspase

in vivo Imaging of Retinal Caspase Activity with the Novel Near Infrared Tracer: 780-VAD-FMK

Jeffrey A Jamison1, Gary L Johnson 2

1 Ophthy-DS, Inc., Portage, MI. 2 Seed Intracellular Technologies, Inc. Minneapolis, MN

BACKGROUND 780-VAD-FMK

INTRODUCTION RESULTS

Apoptosis is the primary death pathway in diseases such as AMD, Diabetic Retinopathy, Retinal Detachment, and Glaucoma. Models of these conditions exhibit complications such as extended development of pathology, variability of induced disease, or lack of sensitive in vivo endpoints. This increases drug development costs and limits productivity. A method to evaluate apoptosis in vivo would enhance disease monitoring, provide a quick assay of efficacy, and allow pre-screening of subjects for study inclusion. 780-VAD-FMK is a low molecular weight infrared tracer designed for use in detecting apoptosis, which binds to active caspases and accumulates transiently within the cells. The purpose of this study is to evaluate 780-VAD-FMK for use in ocular disease.

Blue Light Damage (BLD) is a high through put in vivo model used to screen compounds which protect photoreceptor and RPE cells undergoing apoptosis similar to retinal diseases such as Dry AMD, Retinitis Pigmentosa (RP) and retinal detachment. BLD has many hallmarks similar to Geographic Atrophy associated with Dry AMD such as an increase in oxidative stress, the creation of mitochondrial induced reactive oxygen species in the RPE, increased deposition of complement factor C3 and the creation of Carboxyethylpyrrole (CEP) adducts in the retina. Typical end points to evaluate damage and protection are functional such as Electroretinography (ERG) and structural such as Histology and OCT. Following light exposure photoreceptors and RPE cells begin a process of apoptosis, inflammation and phagocytosis lasting 3-5 days resulting in regions of significant retinal thinning similar to Geographic Atrophy . The 3-5 day remodeling process provides and ideal opportunity to evaluate NIR cell permeant tracer 780-VAD-FMK

STUDY DESIGN

ADDITIONAL OBSERVATIONS

During the study it was observed that a low level of non-specific binding of a control dye occurred in some potentially apoptotic regions. Because the control dye has no caspase localization factor, there can be no mechanistic targeting of apoptotic events with the control. Experimental work is ongoing to determine the relative staining of tracer vs. control and to understand the nature of staining by the control. Neither the control dye or 780-VAD-FMK tracer demonstrated any accumulation in retinas that had no light damage. The light damaged retinas contained a significantly greater amount of 780-VAD-FMK tracer than was present in the control dye study arm. Future work will include optimization of imaging time and dose as well as application to additional disease models.

Pigmented Lewis Rats were exposed to intense blue light on Day 0. On Day 2 non-light exposed and light exposed animals received 100 nM/kg of 780-VAD-FMK intravenously and were imaged using the Heidelberg Spectralis confocal scanning laser ophthalmoscope. Tracer was visualized using ICG angiography mode. OCT was collected on Day 2 and Day 10 following tracer injection.

NHNH

O

O

O

OCH3

O

N O+ N

S

O-

O

O

NH

O

F

C3

A

D C

B

F

E

BACKGROUND BLUE LIGHT DAMAGE

Tracer accumulation corresponds

with start of retinal thinning

Lack of tracer accumulation

corresponds with normal retina

No significant tracer accumulation

in normal or stable damaged retina

Diffuse tracer circulation in

choroid with no accumulation Tracer is present in retinal

Vasculature with no accumulation

Focusing deeper in the retina shows

Tracer accumulation in the superior retina

and better resolution of damaged tissue

Tracer is present in retinal vasculature

with accumulation in the superior

retina localizing to damaged tissue

Blue Light Damage Chamber

Deposition of Caspase 3

2 Days Post BLD

780-Val-Ala NH F

O

O

OH

NH F

O

O

S

OH

Cys-Enzyme

_

780-Val-Ala

HS-Cys-Caspase

NH F

OH

O

O

S

Cys-Enzyme

_

780-Val-Ala

Cys-Enzyme

780-Val-Ala NH

OH

O

O

S

_

+

Cys-Enzyme

780-Val-Ala NH

OH

O

O

S

_

+

Cys-Enzyme

NH

OH

O

O

S

780-Val-Ala

Control NIR Dye

780-VAD-FMK Tracer