Download The effect of combined daunorubicin and triamcinolone

Investigative Ophthalmology & Visual Science, Vol. 33, No. 7, June 1992
Copyright © Association for Research in Vision and Ophthalmology
The Effect of Combined Dounorubicin ond
Triomcinolone Acetonide Treatment on a Refined
Experimental Model of Proliferative Vitreoretinopathy
Edward P. Chen,* Ulrich H. Sreinhorsf,t§ Gregory P. Samsa4§ Peter T. Saloupis,t and Diane L. Harchell*t§
Prior studies have shown that intravitreal daunorubicin (9-15 nmol) and triamcinolone acetonide (2
mg) are effective individually in preventing retinal detachment in experimental proliferative vitreoretinopathy. This report compares the efficacy of the combination of daunorubicin (15 nmol) and triamcinolone acetonide (2 mg) with that of daunorubicin alone in a refined experimental model of proliferative vitreoretinopathy. The degree of retinal detachment in each treatment group was graded, with the
unequivocal absence or presence of retinal detachment used as an indicator of treatment success or
failure. Both treatments (daunorubicin alone and in combination with triamcinolone acetonide) effectively prevented retinal detachment. However, there was no significant difference in the rate of retinal
detachment between the two treatment groups. These results indicate that combination therapy with
daunorubicin/triamcinolone is no more effective at preventing retinal detachment than daunorubicin
alone. Invest Ophthalmol Vis Sci 33:2160-2164, 1992
cacious in preventing retinal detachment in this
model, but to a lesser degree. When injected 24 hr
prior to cell injection, 2 mg triamcinolone acetonide
reduced the rate of retinal detachment from 85%
(control) to 43% (treated).10
The purpose of this study was to determine if combination treatment with daunorubicin and triamcinolone acetonide would be more efficacious than treatment with daunorubicin alone in preventing retinal
detachment. If so, it might be possible to decrease the
concentration of daunorubicin or triamcinolone acetonide to reduce the chance of retinal toxicity. Preliminary results were presented at the May 1991 ARVO
meeting in Sarasota, Florida.
Proliferative vitreoretinopathy (PVR) is a major
complication of retinal surgery. This condition is
characterized by cellular proliferation on the anterior
and posterior surfaces of the retina and along formed
vitreous, resulting in the formation of strands and
membranes as well asfixedretinal folding.12 Mechanical removal of these membranes allows for the treatment of these diseases. However, the clinician often is
faced with the recurrence of proliferation.
Animal models have been developed that can reproduce many of the features of PVR.3"8 One such
model, the refined experimental model of proliferative vitreoretinopathy, involves the injection of
25,000 fibroblasts into an eye that has previously undergone gas-compression vitrectomy.8 We showed
that 15 nmol daunorubicin in this model resulted in a
reduction of retinal detachment from 100% (control)
to 25% (treated) when injected in divided doses 3 d
after cell injection.9 Corticosteroids alone also are effi-
Materials and Methods
Preparation of Cultured Fibroblasts
Dermalfibroblastswere obtained from rabbit rump
explants, prepared under sterile conditions and incubated in 75 cm2 culture flasks (Corning, Wexford,
PA). Primary cultures were maintained in Dulbecco's
modified Eagle's medium with 10% fetal bovine
serum, antibiotics (penicillin sodium, streptomycin
sulfate), and antimycotics (amphotericin B) under a
humidified atmosphere of 5% carbon dioxide in air.
Cells were harvested by incubating them with 3.5 ml
of 0.04% trypsin for 4 min and collecting the cells in
stop media (Dulbecco's modified Eagle's medium
with 10% calf serum plus antibiotics and antimycotics). The dispersed cells were centrifuged at 900 rpm
From the Departments of fOphthalmology, "Cell Biology, and
^Community and Family Medicine Division of Biometry, Duke
University, and §Durham Veterans Affairs Medical Center, Durham, North Carolina.
This work was supported in part by a National Eye Institute Research Grant EY 02903 (DLH), Deutsche Forschungsgemeinschaft
(Bonn, Germany) Grant Ste-418/1-1 (UHS), Veterans Affairs Medical Research Funds, Washington, D.C. (DLH), and Research to
Prevent Blindness, Inc., New York, New York (DLH).
Submitted for publication: September 12, 1991; accepted January 6, 1992.
Reprint requests: Dr. Diane L. Hatchell, Department of Ophthalmology, Box 3802, Duke University Eye Center, Durham, NC
27710.
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COMBINATION THERAPY IN EXPERIMENTAL PVR / Chen er ol
for 10 min and resuspended in 4 ml of phosphate-buffered saline (PBS). The cell count in a 0.1 ml aliquot
was determined and enough PBS was added to
achieve a final concentration of 25,000 per 0.1 ml in
the remainder of the suspension.
Gas Compression Vitrectomy and Gas-Fluid
Exchange
Fifty Dutch-belted pigmented rabbits of both sexes
weighing 2.5-5.0 pounds were anesthetized with 0.5
ml ketamine and 0.1 ml xylazine. Their pupils were
dilated with 5.0% phenylephrine and 0.25% tropicamide prior to all procedures and photographs. All experimental procedures conformed to the ARVO Resolution of the Use of Animals in Research.
The technique of gas-mediated vitreous compression as part of the refined experimental model of PVR
has been previously described.811 Transcleral cryolesions were placed to create an area where repeated
injections could be made. Ten days after cryopexy,
perfluoropropane gas (0.4 ml) was injected. Two days
later, the gas had expanded to fill 80%-90% of the
vitreous cavity. A syringe with a 30 G needle was inserted inferiorly, and approximately 0.2-0.3 ml of
sterile Ringer's lactate solution was injected. Gas
escaped readily from the hole when the needle was
withdrawn. This process of slow injection and gas
escape was repeated until gas could no longer be seen
in the eye. The entry site then was closed with a preplaced 7-0 Vicryl (Ethicon, Somerville, NJ) suture.
Intravitreal Injections of Fibroblasts, Daunorubicin,
and Triamcinolone Acetonide.
Ten days after gas-fluid exchange, the eye was proptosed and a 27 G needle was inserted through the
sclera and retina 4 mm posterior to the corneoscleral
junction in the superotemporal quadrant under stereomicroscopic control. With the bevel of the needle
directed downward, 25,000 tissue-cultured homologous dermal fibroblasts suspended in 0.1 ml sterile
PBS were injected over the optic nerve and medullary
rays. The animals were immediately placed on their
backs for 1 hr to allow the cells to settle over the vascular wings of the retina.8
The rabbits then were randomized into three
groups. Group 1 (n = 18) received one intravitreal
injection of triamcinolone acetonide, 2 mg/0.1 ml
0.005% tyloxapol, 24 hr prior to cell injection and 10
nmol daunorubicin/0.1 ml PBS followed by a second
injection of 5 nmol daunorubicin/0.05 ml PBS 4 hr
thereafter on the third day after cell injection. Group
2 (n = 16) received daunorubicin at the same dosages
and times as well as the triamcinolone vehicle. Group
3 (n = 16) received both drug vehicles. Injection of the
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drugs and vehicles was performed identically to that
of fibroblast injection.
Clinical Examination
Each eye was examined by indirect ophthalmoscopy and followed by fundus drawings and fundus
photography on days 0, 3, 7, 14, and 28 after fibroblast injection. All rabbits were killed on day 28 with
an overdose of 1.0 ml pentobarbital sodium via ear
vein infusion.
Grading of Proliferative Vitreoretinopathy
The progression of proliferative vitreoretinopathy
was graded using the classification system of Hida et
al.12 Stage 0 showed no abnormalities in the fundus.
Stage 1 showed surface wrinkling of the medullary
wing, resulting in a beaten metal appearance. Stage 2
showed mild puckering and small focal contractions
on the nonelevated wings. Stage 3 showed severe
puckering and contraction of the whole medullary
wings. Stage 4 showed elevated puckering caused by a
vitreous strand. Stage 5 showed partial retinal detachment involving one medullary wing. Stage 6 showed
low retinal detachment involving both wings with the
avascular retina attached. Stage 7 showed total retinal
detachment with a closed funnel appearance. The unequivocal absence (grades 0-4) or presence (grades
5-7) of retinal detachment was used as the indicator
of success or failure of combination therapy and treatment with daunorubicin alone. The results were subjected to statistical analysis using the Fisher's exact
test.
Observation of Neovascularization
All eyes that progressed to stage 7 retinal detachment were observed and documented for the presence
of concomitant neovascularization.
Results
Proliferative Vitreoretinopathy
All animals receiving only drug vehicles (n = 16)
developed stage 7 retinal detachment (100%) by day
28 (Figure 1). Seven of the 16 animals receiving daunorubicin alone developed retinal detachment
(43.8%) by day 28. Of these failures, two eyes were
stage 5, two were stage 6, and three were stage 7. Five
of the 18 animals receiving both daunorubicin and
triamcinolone acetonide developed retinal detachments (27.8%) by day 28. Of these failures, two eyes
had stage 5 detachments, one had a stage 6 detachment, and two eyes had stage 7 detachments. The
overall distribution of final stage for all eyes at day 28
is shown in Table 1.
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / June 1992
Control
DNM
DNM/TA
Fig. 1. Graph comparing the percentage of eyes with presence of
retinal detachment in control and experimental groups at day 28.
The rate of retinal detachment at day 28 was significantly less in both treated groups compared to controls (P < 0.0001, Fisher's exact test). There was no
significant difference between the experimental
groups in prevention of proliferative vitreoretinopathy (risk ratio = 13.9; 95% confidence interval
= 0.58-3.29).
Neovascularization
Twelve of the 16 stage 7 control eyes developed
concomitant neovascularization as well. All three of
the stage 7 eyes in the daunorubicin group developed
neovascularization (100%), whereas neither stage 7
eye in the combination therapy group developed neovascularization (Figures 2, 3). Statistical analysis was
not performed on these observations because of the
small number of rabbits with stage 7 eyes.
Discussion
Although treatment with daunorubicin alone and
in combination with triamcinolone acetonide led to a
significant decrease in the rate of retinal detachment
in experimental proliferative vitreoretinopathy compared to controls, combination treatment with daunorubicin and triamcinolone acetonide was shown to be
no more effective than treatment with daunorubicin
alone. There was no statistically significant difference
between the treatment groups regarding the rate of
retinal detachment (43.8% in the daunorubicin group
vs. 27.8% in the combination group). The 95% confidence interval for the difference between these two
proportions is 16 ± 32% (mean ± standard error of the
mean). This width indicates that our experiment did
not have the precision to detect subtle differences in
outcome between these two groups (ie, we could detect differences in the order of 28% vs. 60%, which is
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Vol. 33
32% on the absolute scale, but we could detect no
smaller differences). We would have needed approximately 60 rabbits per group to have had the statistical
power to detect a difference in response rates on the
order of 44% versus 28%. Because this was prohibitively expensive, we stopped the experiment. It is disappointing that our results did not show better efficacy with the drugs in combination. We had hoped to
be able to next decrease the concentration of daunorubicin because daunorubicin has a low therapeutic index. Wiedemann et al13 found that doses above 27
nmol were toxic to the retina, as evidenced by the
presence of retinal holes and retinal detachments.
Santana et al14 demonstrated photoreceptor outer segment damage, by light and electron microscopy, in
dosages as low as 15 nmol, whereas even at the therapeutic dose of 9 nmol, outer segment abnormalities
were observed by electron microscopy. Triamcinolone acetonide (1 mg), on the other hand, does not
cause ocular toxic effects as assessed by slit-lamp examinations, ophthalmoscopy, light and electron microscopy, electroretinography, or intraocular pressure
measurements.15
Many investigators previously have proven daunorubicin9'1316 and triamcinolone acetonide101718 to be
individually effective in preventing retinal detachment in experimental models of proliferative vitreoretinopathy. However, no studies have examined the
efficacy of a combination of these two agents. The
purpose of this experiment was to combine daunorubicin with a substance known to be less toxic to the
retina and with a different mechanism of action to
further decrease the rates of retinal detachment.
The cytostatic effects of daunorubicin, an anthracycline antibiotic, have been proposed to depend on a
number of mechanisms involving DNA binding, free
radical formation, ion-pump inhibition, metal ion
chelation, and cell membrane disruption.1920 Preferential binding of these agents to DNA instead of RNA
Table 1. Stages of proliferative vitreoretinopathy of
Hida et al.12 in control and treated eyes on day 28
(number of eyes)
Control
Daunorubicin
alone
Daunorubicin and
triamcinolone
acetonide
0
1
2
3
4
5
6
7
0
0
0
0
0
0
0
16
2
0
2
3
2
2
2
3
2
1
0
1
9
2
1
2
Total
16
16
18
Stage
(Hidaetal.14)
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COMBINATION THERAPY IN EXPERIMENTAL PVR / Chen er al
also has been shown to lead to mutagenic, teratogenic,
and carcinogenic effects in living organisms.
Daunorubicin has been proven to be efficacious in
preventing retinal detachment in experimental proliferative vitreoretinopathy. Wiedemann et al13 demonstrated that a dose of 9 nmol per eye reduced the incidence of retinal detachment in experimental proliferative vitreoretinopathy from the 75% seen in controls
to 25% after 28 d, whereas doses between 5 and 15
nmol per eye were effective in preventing retinal detachment. In the refined experimental model of proliferative vitreoretinopathy,8 Khawly et al9 showed that
daunorubicin, in divided doses of 10 nmol followed
by 5 nmol 4 hr later on the third day after cell injection, reduced the rate of retinal detachment from
100% to 25%. Daunorubicin was not effective as a
single 15 nmol dose. In this case, the rate of retinal
detachment was reduced from 100% to 73%.
Steroids exert their antiproliferative effect by inhibiting DNA, RNA, and protein synthesis as well as
altering cell membrane permeability.21"24 Triamcinolone acetonide inhibits fibroblast growth21'22 and mitotic activity in cells in tissue culture.23-24 Tano et al17
injected 250,000 fibroblasts into rabbit eyes and
found that simultaneous injection of 1 mg triamcinolone acetonide reduced the percentage of traction retinal detachment from 84% to 20% and the percentage
of neovascularization from 58% to 0% in eyes receiving triamcinolone acetonide compared with eyes receiving only the drug vehicle. Chandler et al18 reduced
the rate of retinal detachment from 90% to 56% with 2
mg triamcinolone acetonide in the refined experimental model of proliferative vitreoretinopathy. An improvement in the efficacy in the same model was dem-
Fig. 2, Fundus photograph of rabbit showing a stage 7 retinal
detachment with the presence of neovascularization.
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Fig. 3. Fundus photograph of rabbit showing a stage 7 retinal
detachment with the absence of neovascularization.
onstrated when the drug was injected 24 hr prior to
cell injection.10 Antoszyk et al later demonstrated that
in an experimental model of preretinal neovascularization in the rabbit,25 pretreatment with 2 mg triamcinolone acetonide reduced the incidence of severe
neovascularization from 100% to 14%,26 whereas
treatment with 2 mg triamcinolone acetonide 3 d
after hyaluronidase and cell injection reduced the rate
of severe neovascularization from 100% to 54%.27
The rate of retinal detachment in eyes receiving daunorubicin alone was found to be 43.8% in our experiments. This rate is slightly higher than that shown by
Khawly et al,9 who demonstrated a 25% rate of retinal
detachment in groups treated with daunorubicin.
This discrepancy may be explained by a difference in
the experimental protocol. To maintain identical
conditions among all experimental groups, it was necessary for us to inject the daunorubicin group with the
triamcinolone acetonide vehicle 24 hr prior to cell
injection so a valid comparison between the two treatment groups could be made. This extra injection constitutes an additional breakdown of the blood-retinal
barrier. Thus, conditions in the two experiments were
not the same.
Although not the main focus of the present investigation, we were curious about whether triamcinolone
acetonide would decrease the rate of neovascularization in stage 7 retinal detachments, as shown in previous reports. We observed that in eyes receiving
triamcinolone acetonide (ie, the combination group),
there was no evidence of neovascularization in eyes
with stage 7 retinal detachment, whereas in eyes that
did not receive triamcinolone acetonide, neovascularization was observed in the majority of eyes with stage
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / June 1992
7 retinal detachment (all three eyes in the daunorubicin group and 12 of the 16 control eyes). Because we
did not perform statistical analysis on this data, these
results do not confirm previous investigations by Antoszyk et al but are consistent with them.25"27
Key words: daunorubicin, triamcinolone acetonide, proliferative vitreoretinopathy, PVR, retina, vitreous.
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