1. 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.
NH
NH
O
O
O
OCH3
O
N O+ N
S
O-
O
O
NH
O
F
C3
A
DC
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