|Year : 1994 | Volume
| Issue : 1 | Page : 31-35
Experimental inhibition of proliferative vitreoretinopathy in retinal detachment using daunorubicin
Atul Kumar, HK Tewari, DP Bathwal, PK Khosla
Dr. Rajendra Prasad Centre for Ophthalmic Sciences, AIIMS, New Delhi, India
Dr. Rajendra Prasad Centre for Ophthalmic Sciences, AIIMS, Ansari Nagar, New Delhi 110 029
Source of Support: None, Conflict of Interest: None
Proliferative vitreoretinopathy (PVR) remains the most common cause of failure in retinal detachment surgery. Surgical procedures for its repair entails complex and extensive instrumentation besides technical skill. The success rate varies widely with high incidence of redetachment. Keeping this in view, we evaluated the role of intravitreal daunorubicin as an anti-mitiotic agent in the inhibition of PVR. Our study concluded that 5 micrograms of intravitreal daunorubicin effectively inhibited PVR in the rabbit eye and the dosage was safe and nontoxic. The half-life of the drug was determined to be about 140 minutes, suggesting a prolonged intravitreal concentration sufficient to prevent fibroblast proliferation
Keywords: Proliferative vitreoretinopathy - Retinal detachment - Anthracycline antibiotics - Daunorubicin.
|How to cite this article:|
Kumar A, Tewari H K, Bathwal D P, Khosla P K. Experimental inhibition of proliferative vitreoretinopathy in retinal detachment using daunorubicin. Indian J Ophthalmol 1994;42:31-5
The incidence of proliferative vitreoretinopathy (PVR) in rhegmatogenous retinal detachment (RD) is 5 to 10%. It is characterised by migration, metaplasia, proliferation of retinal pigment epithelium (RPE), glial cells, and macrophages leading to formation of fibrocellular membranes on both surfaces of the detached retina, which on contraction leads to distortion or detachment of the retina. Though recent advanced procedures of vitrectomy have remarkably improved the surgical prognosis, both anatomical and functional failure rates remain considerable.
Various drugs have been used in the past to prevent PVR. Corticosteroids and 5-fluorouracil (5-FU) have been demonstrated to significantly inhibit PVR,  but require repeated administration and are not without side effects. Binder et all have demonstrated that 1 to 5 mg of 5-FU reduced tractional RD in the rabbit eye from 75 to 3% after 4 weeks but noticed retinal toxicity viz. narrowing of vessels and haemorrhage, early retinal oedema, degeneration of RPE, swelling and loss of axons and mitochondria in outer plexiform layer. The effective dose of 5-FU for inhibition of PVR was determined to be 0.5 mg injected intravitreally every 24 hours for 7 days.'
Doxorubicin and Daunorubicin are anthracycline antibiotic drugs isolated from the soil fungus Streptomyces peucetius var caesius. Wiedemann et al [l] have shown that 9 nmol of daunorubicin reduced PVR in RD by 50%. They also found that 500 nM of the drug for 5 hours or 1000 nM for 1 hour completely inhibited colony forming units (CFU) in vitro. Anthracycline antibiotics are unique in action as they are effective in all phases of cell cycle; hence a single intravitreal administration is sufficient to inhibit PVR.
We hereby report the toxic effects, clearance and in vivo inhibition of daunorubicin (DRB) following intravitreal administration.
| Materials and methods|| |
Thirty-six rabbits were taken into our study after ruling out any ocular pathology including the presence of any cataract or retinal detachment.
Determination of Safe Ocular Dose
Forty eyes of 20 rabbits were taken into this study. Rabbits were anaesthetised by injecting pentobarbitone 30 mg/kg intraperitoneally. Their pupils were dilated with 1% tropicamide and 10% phenylephrine topically. These eyes were divided into 2 groups.
Group I (Controls): 20 rabbit eyes taken as control were injected with 0.1 ml of balanced salt solution (BSS) intravitreally through pars plana route.
Group II (Treated) 20 rabbit eyes were injected with various intravitreal concentrations of DRB in 0.1 ml of BSS solution (5 eyes each with 5, 6.0, 7.5, and 12.5 µg DRB). Fundus examination, including photography, and electroretinography (ERG) were carried out in all these eyes.
Fundus examination was carried out using indirect ophthalmoscope. Fundus photographs were taken on 0, 1, and 7 days post-intravitreal injection. Electroretinographic recordings were performed with the Amplaid MK-15 Electro diagnostic system on 0, 1, and 7 days and a-wave and b-wave amplitude and implicit time of the b-wave were compared over this time period with the fellow control eye. Electron microscopy was carried out with various concentrations of the drug using the Philips CM-10 TEM system, after enucleation.
Elimination of DRB from the Vitreous
Twelve rabbit eyes (six rabbits) were included for this part of the study. Rabbits were anaesthethised with intraperitoneal pentobarbitone 30 mg/kg and topical anaesthesia with 4% xylocaine. Five microgram of DRB in 0.1 ml of BSS solution (determined as nontoxic) was taken in a tuberculin syringe and injected transconjunctivally through pars plana route into the midvitreous using a 26-gauge needle. The direction of the needle was kept posterior to avoid damage to the lens. The corneal button was excised at the limbus to avoid bleeding and to prevent vitreous contamination with blood. The lens was removed by applying pressure at the limbus at the 6 o'clock position and the vitreous was aspirated with a 5-ml syringe at various time intervals-immediately, and after 30, 60, 90, 120, and 150 minutes post-injection. Extracted vitreous was then mixed with an equal volume of 4:1 mixture of 0.1 M phosphoric acid and acetonitrile, and centrifuged for 1 hour in a REMI laboratory centrifuge (Model-R 4C). The supernatant containing the drug was then aspirated with a pipette and transferred to a cuvette. The amount of the drug in the supernatant was calculated with a spectrofluorometer using an excitation wavelength of 470 nm and an. emission wavelength of 585 nm.
Daunorubicin standards carried out through the extraction procedures were used to calculate the amount of the drug in the supernatant. The half-life of the drug was calculated as follows:
Slope (emission rate constant) = 0.693 / t 1
= log C 1 - log C 2
t 2 - t i
C, and C 2 being concentrations of the drug at various time intervals, t i and t 2 .
Inhibition of Fibroblasts (in vivo)
The third part of the study involved injection of a suspension of live, homologous fibroblasts into the rabbit eye and studying the effect of intravitreal 5 gg daunorubicin on fibroblasts. Twenty rabbit eyes (10 rabbits) were included in this part of the study. One eye of each animal acted as test eye, and the fellow eye as control.
Homologous live dermal fibroblasts were seeded in tissue culture flasks in RPMI 1640 medium containing 10% foetal serum and antibiotics. After 4 to 5 days the cells formed a monolayer along the sides of the flask and produced sufficient number of cells for intravitreal injection. Cells were then removed with phosphate-buffered saline (PBS) and counted in a counting chamber.
Twenty-five thousand live fibroblast cells in 0.1 ml were injected through the pars plana route into the rabbit eye; subsequently daunorubicin (5µg) was also injected into the same eye through the pars plana route. The fellow eye of the rabbit served as control and each animal was followed up on 3rd, 7th, 14th, and 28th day.
The evaluation consisted of indirect ophthalmoscopy, fundus photography, and electroretinography.
| Results|| |
Various doses of daunorubicin 5, 6.0, 7.5 and 12.5 gg of daunorubicin were injected intravitreally. Fundus examination was done with the indirect ophthalmoscope and the photographs were taken with Topcon TRC 50X Fundus camera using 400 ASA 35 mm film on 0, 1, and 7 days. Retinal vasculitis and haemorrhages were noticed with 7.5 gg of the drug [Figure - 1].
Electroretiographic recordings of a-wave amplitude, b-wave amplitude and implicit time were taken on 0, 1, and 7 days which showed statistically significant depression of b- wave amplitude with 6 gg and higher concentrations of the drug [Table - 1][Table - 2][Table - 3].
Transmission electron microscopy revealed vacuolation in all retinal layers indicating hydrophilic degeneration due to drug toxicity. This was extremely marked with 7.5 µg and higher concentrations of the drug. Vacuolation was also observed, though minimal, with 6 ug of the drug [Figure - 2][Figure - 3].
Spectrofluorometric readings showed that elimination of the drug from the vitreous follows first order kinetics and the half-life of the drug was 140 minutes.
Definite fibroblast inhibition was observed when 5 ug daunorubicin was injected with 25,000 live homologous fibroblasts, while PVR changes occurred in the control eye where only fibroblasts were injected [Figure - 4][Figure - 5].
| Discussion|| |
Daunorubicin is an anthracycline antibiotic used primarily for the treatment of acute leukemias. This drug has been shown to be one of the most effective intraocular antiproliferative agents for the treatment of proliferative vitreoretinopathy.  The exact mechanism of action of anthracyclic antibiotics including daunorubicin is multifactorial. It intercalates with DNA, thereby inhibiting DNA and RNA synthesis, causes DNA strand scission due to inhibition of topoisomerase II enzyme and free radical formation. Daunorubicin also generates semiquinone free radicals and superoxide radical through a Cytochrome P-450 mediated reductive process .  Its action being independent of the cell cycle, only short exposure times are required.'
A drug appropriate for the control of PVR must possess two qualities: it has to inhibit cell proliferation effectively, and it must do this without intolerable toxicity to the retina or other ocular structures. The effectiveness of a drug depends upon its pharmacokinetic and pharmacodynamic properties. The desired pharmacokinetic properties include suitability for local injection into the vitreous as this results in highest possible concentration at the target site, thus eliminating, or at least reducing systemic toxicity. Also, the drug must remain long enough to affect the proliferation of cells during a sensitive period of their cell cycle.
We performed retinal examination and fundus photography on 0, 1, and 7 days after injecting various concentrations of DRB intravitreally. With 5 µg we observed no change in the fundus while with higher doses, i.e. with 6 µg, vasculitis was observed and with 7.5 and 12.5 gg associated retinal haemorrhage was noticed.
ERG recordings were taken and comparison of awave amplitude, b-wave amplitude, and implicit time were done on 0, 1, and 7 days after injecting various intravitreal concentrations of DRB. There was no change in the ERG recordings between the controls and the 5 µg group but there was a significant change in b-wave amplitudes with 6, 7.5, and 12.5 µg of the drug. In other words, the b-wave amplitude values on ERG were strikingly affected whenever higher concentrations of DRB were injected. We definitely feel that the significant change in the b-wave amplitude is directly related to retinal toxicity. Our observation was in accordance with that of Barrada et a1  who also found that 5 µg of the drug to be safe and nontoxic on electroretinography.
Transmission electron microscopy is an extremely effective investigation to detect subtle changes in the retinal layers.  In our study, electron microscopy disclosed vacuolation in all retinal layers, extremely marked with 7.5 µg and higher doses of the drug, suggesting definite retinal drug toxicity. Some evidence of vacuolation was also observed with 6 µg, but the retinal layers seemed unaffected with 5 gg daunorubicin. This is in slight contradiction to previous reports by Weidemann et al,  where it has been shown that 9 nmol (approximately 6.0 µg) of daunorubicin did show some photoreceptor outer segments.
Regarding the clearance of the drug from the vitreous, we determined that aspirated vitreous volumes taken for assay were between 1.2 to 1.5 ml. The half-life of the drug estimated on spectrofluorometry after its intravitreal injection was 140 minutes."' As the drug follows first order kinetics, the total duration for complete removal would thus be approximately 4 to 5 half-lives, i.e. about 560 to 700 minutes; hence effective and prolonged intravitreal concentrations were therefore achieved.
The in vivo PVR in the rabbit eye was observed after creating an animal model by first causing a posterior vitreous detachment using 0.5 to 0.8 ml sterile air, and subsequent injection of fibroblasts (25,000 cells) which then grew along the epiretinal surface to induce
PVR changes. Injection of the reasonably nontoxic 5 µg daunorubicin showed none of the changes suggestive of proliferative vitreoretinopathy even after one month of follow-up, thus proving its efficacy. This in vivo inhibition of PVR in the rabbit eye using daunorubicin has not been previously reported. To the best of our knowledge, this is the first study where experimental in vivo inhibition of PVR was achieved using 5 µg of daunorubicin.
In a clinical study involving 13 eyes with advanced traumatic proliferative vitreoretinopathy, Wiedemann et a1  have demonstrated the effectiveness of 7.5 µg/ ml of intravitreal daunorubicin in the inhibition of PVR, wherein 7.5 µg/ml of daunorubicin was infused over a ten-minute period. Our work has demonstrated that 5 µg of this drug is sufficient to inhibit PVR, has a relatively long half-life and exhibits no retinal toxicity.
In view of the above facts, we feel that 5 gg of daunorubicin is a safe and nontoxic dose in the inhibition of PVR. Clinical trials using intravitreal daunorubicin in selected eyes with active primary proliferative vitreoretinopathy, and also after PVR surgery to prevent reproliferation of epiretinal membranes, would be reasonably justified in view of our experimental findings.
| Acknowledgement|| |
This study was supported by S.Y.S. Project, Dept. of Science & Technology (DST), Mehrauli Road, New Delhi.
| References|| |
Chandler DB, Quansah FA, Hida T, et al. A refined experimental model for proliferative vitreoretinopathy Graefes Arch Clin Exp Ophthalmol. 224:86-91, 1986.
Blumenkranz MS, Ophir A, Clafin AJ, et al. Fluorouracil for the treatment of massive periretinal proliferation. Am J Ophthalmol. 94:458-567, 1982.
Peyman GA, Greenberg D, Fishman GA, et al. Evaluation of toxicity of intravitreal antineoplastic drug. Ophthalmic Surg. 15:411-413, 1984.
Blumenkranz MS, Hernandez E, Ophir A, et al. Fluorouracil: New applications in complicated retinal detachment for an established metabolite. Ophthalmology. 91:122-130, 1984.
Sunalp MA, Wiedemann P, Sorgenete N, et al. Effect of adriamycin on experimental proliferative vitreoretinopathy in the rabbit. Exp Eye Res. 41:105-115, 1985.
Binder S, Sorpik C, Paroussi P, et al. Inhibition of experimental intraocular proliferation and retinal detachment by various drugs. In: Blankenship GW, Strip M, Binder S (eds). Basic and Advanced Vitreous Surgery Padoval, Springer Verlag Livina Press, pp. 407-414,1986.
Stern WH, Lewis GP, Erickson PA, et al. Fluorouracil therapy for proliferative vitreoretinopathy after vitrectomy. Am J Ophthal. 96:33-42, 1983.
Wiedemann P, Kirmani M, Sorgente N, et al. Control of experimental massive periretinal proliferation by daunomycin dose-response relation. Graefes Arch Clin Exp Ophthalmol. 220:233-235, 1983.
Kirmani M, Santana M, Sorgente N, et al. Antiproliferative drugs in treatment of experimental proliferative vitreoretinopathy. Retina. 3:269, 1983.
Wiedemann P, Sorgente N, Bekhor C, et al. Daunomycin in the treatment of experimental proliferative vitreoretinopathy. Invest Ophthalmol. 26:719-725, 1985.
Wiedemann P, Leinung C, Hilgers RD, et al. Daunomycin and silicone oil for the treatment of proliferative vitreoretinopathy. Graefes Arch Clin Exp Ophthalmol. 229:150-152, 1991.
Calabresi P, Chabner BA. Antineoplastic agents. In: Gilman AG, Ball TW, Niles AS, Taylor P(eds). Goodman and Gilman's. The pharmocological basis of therapeutics. Vol. II, Maxwell Macmillan Pergamon Publishing Corpn. pp. 1209-1263, 1990.
Barrada A, Peyman GA, Greenberg D, et al. Toxicity of antineoplastic drugs in vitrectomy infusion fluids. Ophthalmic Surg. 14:845-847, 1983.
Wiedemann P, Lemmen K, Schmiedl R, et al. Intraocular daunorubicin for the treatment and prophylaxis of traumatic proliferative vitreoretinopathy. Am J Ophthalmol. 104:10-14, 1987.
[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5]
[Table - 1], [Table - 2], [Table - 3]