Rabbits and monkeys in this study were obtained and cared for in
accordance with all applicable federal and state guidelines for animal
care. The use of animals adhered to the ARVO Statement for the Use of
Animals in Ophthalmic and Vision Research.
Rabbits were treated with either ISIS 2922 (33 or 82 μg/eye) or ISIS
13312 (36 or 90 μg/eye) by single intravitreal injection (0.05 ml) to
compare ocular kinetics. Intravitreal injections were performed using a
30-gauge needle placed 2 to 3 mm behind the limbus after a
povidone-iodine rinse of the eye. The doses were chosen to yield
initial estimated vitreal concentrations of 4 or 10 μM
oligonucleotide at the high and low doses, respectively, assuming a
1.0-ml vitreal volume. The greater amount of ISIS 13312 administered
reflected the slightly greater molecular weight of this
oligonucleotide. Animals were killed and eyes collected at the
indicated time point for measurement of either vitreal or retinal
concentrations of oligonucleotide.
Another group of rabbits were treated with ISIS 2503 (82 μg/eye) or
ISIS 13920 (90 μg/eye) by single intravitreal injection (0.05 ml) to
compare ocular tissue distribution. The targeted initial vitreal
concentration in these animals was 10 μM, and injections were
performed as for ISIS 2922 and ISIS 13312. At 2, 4, 10, or 30 days
after injections, animals were killed and eyes collected for
immunohistochemical analysis. The lens was removed to facilitate
fixation and eyes were placed in formalin-paraformaldehyde solution.
After fixation, eyes were embedded in paraffin and cut to 4-μm
sections. Sections were deparaffinized, blocked with normal donkey
serum, incubated for 1 hour with the anti-oligonucleotide monoclonal
antibody (diluted at 1:2000), rinsed, and incubated with donkey
anti-mouse IgG-peroxidase conjugate diluted 1:100 for 1 hour.
Peroxidase activity was visualized using 3,3′-diaminobenzidine
substrate, and slides were counterstained with hematoxylin and mounted
in a permanent mounting medium. It was necessary to use ISIS 2503 and
ISIS 13920 for immunolocalization after intravitreal injection, because
the antibody used in this study does not bind to ISIS 2922 or ISIS
13312.
Young adult cynomolgus monkeys were treated with 125, 250, or 500μ
g/eye ISIS 13312 by single and repeated intravitreal injections
(0.05 ml) to study accumulation over time and with increasing dose.
Intravitreal injections were performed using a 30-gauge needle placed 2
to 3 mm behind the limbus after a povidone-iodine rinse of the eye. The
doses were chosen to yield initial estimated vitreal concentrations of
10, 20, or 40 μM oligonucleotide, assuming a 1.1-ml vitreal volume.
Animals were killed and eyes collected at the indicated time point for
measurement of either vitreal or retinal concentrations of
oligonucleotide.
Doses are reported in micrograms, but were administered to achieve
initial vitreal concentrations of oligonucleotide (i.e., 4–40 μM, as
indicated). Based on differences in molecular weight, a slightly
greater amount of the 2′-MOE second-generation oligonucleotides was
administered.