OKG-0301, a Novel Ribonuclease, Demonstrates Antiviral Activity

against Adenovirus in the Ad5/NZW Rabbit Ocular Model.E. G. Romanowski, K. A. Yates, R. M. Q. Shanks, J. E. Romanowski, R. P. Kowalski

The Charles T. Campbell Ophthalmic Microbiology Laboratory

1. 25 µM OKG-0301 and 2.5 µM OKG-0301

demonstrated significant antiviral efficacy

compared with the saline control in the

Ad5/NZW rabbit ocular model.

2. The antiviral efficacy of the 25 µM OKG-0301

group was similar to that of the positive

antiviral control, 0.5% cidofovir.

3. OKG-0301 appears to be a promising

candidate for a topical antiviral for adenoviral

ocular infections and further development is

indicated.

Conclusions

ResultsIntroduction

Adenovirus (Ad) ocular infections

(epidemic keratoconjunctivitis [EKC],

follicular conjunctivitis, and pharyngeal

conjunctival fever) are the most common

ocular viral infections worldwide1. At

present there is no FDA approved antiviral

for the treatment of these infections.

A novel approach to antiviral

development is the use of ribonucleases,

which are enzymes that degrade RNA.

OKG-0301 is a novel ribonuclease that can

enter host cells and preferentially degrade

viral tRNA leading to an inhibition of protein

synthesis. It has been previously shown to

have antiviral activity against HIV2.

UPMC Eye Center

Ophthalmology & Visual Sciences Research Center

The Eye & Ear Institute

Department of Ophthalmology

University of Pittsburgh School of Medicine

Pittsburgh, PA

Website: http://eyemicrobiology.upmc.com

Email: romanowskieg@upmc.edu

3614 - B0173

The goal of the current study was to

evaluate the anti-adenoviral efficacy of

topical OKG-0301 in the Ad5/NZW rabbit

ocular model.

Goals of the Current Study

Experimental Drugs – 25 µM and 2.5 µM OKG-0301

(OKG) were prepared in IV saline from 1 mg vials of

stock drug provided by Okogen. 0.5% Cidofovir (CDV)

was prepared in IV saline from the 7.5% injectable form

of cidofovir (Cidofovir Injection, [Heritage

Pharmaceuticals Inc., Eatontown, NJ]) and served as the

positive antiviral control. IV saline (0.9% Sodium

Chloride Injection USP [Baxter Healthcare Corp.

Deerfield, IL]) served as the negative control (CON).

Virus and Cells – A clinical ocular isolate of adenovirus

type 5 (Ad5) was used in the current study. A549 human

lung carcinoma cells were used to prepare the virus

stock and for the determination of ocular viral titers.

Animals – 1.1 – 1.4 kg female New Zealand White rabbits

were obtained from Charles River Oakwood rabbitry. All

animal studies conformed to the ARVO Statement on the

Use of Animals in Ophthalmic and Vision Research.

University of Pittsburgh IACUC approval was obtained

and institutional and federal guidelines regarding animal

experimentation were followed.

Antiviral Efficacy Study Experimental Design – This

study was performed using a total of 38 rabbits.

Following appropriate systemic and topical anesthesia,

NZW rabbits were inoculated with 50 µl (1.5 x 106

PFU/eye) of Ad5 in both eyes after 12 cross-hatched

strokes of a #25 sterile needle. Twenty-four hours later,

rabbits were randomly assigned to one of four topical

treatment groups:

1) 25 µM OKG – 8X/day x 9 Days (n = 9)

2) 2.5 µM OKG – 8X/day x 9 Days (n = 10)

3) Saline – 8X/day x 9 Days (n = 10)

4) 0.5% CDV – 2X/day x 7 Days (n = 9)

Rabbits were treated topically in both eyes according to

the above treatment regimens. Ocular swabbing to

recover adenovirus from the tear film and corneal and

conjunctival surfaces was performed on days 0, 1, 3, 4,

5, 7, 9, 11, and 13 after inoculation and frozen at -80o C

pending plaque assay.

Determination of Ocular Viral Titers (Plaque Assay) - The

ocular samples to be titered were thawed, diluted, and

inoculated onto A549 cell monolayers. After 7 days

incubation, the cells were stained with 0.5% gentian

violet, and the number of plaques counted. The viral

titers were then calculated, and expressed as plaque

forming units per milliliter (PFU/ml).

Statistical Analysis - Ocular titer data was analyzed

using Kruskal-Wallis ANOVA with Duncan’s multiple

comparisons and Fisher’s Exact Test (True Epistat).

Significance was established at the p < 0.05 confidence

level.

Methods

Financial Support

Okogen, Inc.; NIH Core Grant EY08098

References1. Gordon JS, Aoki K, Kinchington PR. Adenovirus keratoconjunctivitis. In: Pepose JS, Holland

GN, Wilhelmus KR, eds. Ocular Infection & Immunity. St Louis: Mosby; 1996:877-894.

2. Saxena SK, Gravell M, Wu Y, Mikulski SM., Shogen K, Ardelt W, Youle RJ. Inhibition of HIV-1

production and selective degradation of viral RNA by an amphibian ribonuclease. J. Biol. Chem.

1996;271:20783-20788.

Figure 3 demonstrates the Median Ocular Viral Titers over the course of the

study. Significant differences (P = 0.05 K-W) were demonstrated on:

Day 3 (CDV = 2.5 µM OKG = 25 µM OKG < CON)

Day 4 (25 µM OKG = CDV = 2.5 µM OKG < CON)

Day 5 (CDV = 25 µM OKG = 2.5 µM OKG < CON)

Day 7 (25 µM OKG < CDV < 2.5 µM OKG < CON)

Day 11 (25 µM OKG = CDV = 2.5 µM OKG < CON).

Figure 1 depicts the crystallographic structure of OKG-0301 (rainbow) in

complex with RNA (green). Rendered from PDB entry 2I5S using PyMol.

Illustration courtesy of author Opabinia regalis and the GNU Free

Documentation License. (https://commons.wikimedia.org/wiki/File:2I5S.png).

Figure 2 demonstrates the number of Ad5 Positive Cultures per Total over the

course of the study. Significant differences (P < 0.03 FET) were demonstrated on:

Day 5 (CDV < CON)

Day 7 (25 µM OKG = CDV < CON; 25 µM OKG < 2.5 µM OKG)

Day 9 (25 µM OKG < CON)

Day 11 (25 µM OKG = CDV = 2.5 µM OKG < CON).

Median Ad5 Ocular Titers

Days0 1 3 4 5 7 9 11 14

Lo

g1

0 P

FU

/ml

100

101

102

103

104

25 uM OKG

2.5 uM OKG

CON

CDV

Figure 4 demonstrates the median Duration of Shedding. Significant differences

(P = 0.05 K-W) were demonstrated among the groups:

(25 µM OKG = CDV < 2.5 µM OKG < CON).

Days

0 2 4 6 8 10 12 14

Median Duration of Ad5 Shedding

CON

CDV

25 M OKG

2.5 M OKG

Figure 5 demonstrates a patient with acute adenoviral conjunctivitis.

Ad5 Positive Cultures

Days

0 1 3 4 5 7 9 11 14

Pe

rcen

t P

os

itiv

e C

ult

ure

s

0

25

50

75

10025 uM OKG

2.5 uM OKG

CON

CDV