Download Suppression of laser-induced choroidal neovascularization

Suppression of Laser-Induced Choroidal
Neovascularization by Oral Tranilast in the Rat
1,2
Yasuo Takehana, Torn Kurokawa, Tsuyoshi Kitamura, Yoshimi Tsukabara,
Satoshi Akahane,1 Makio Kitazawa,1 and Nagahisa Yoshimura2
PURPOSE. TO determine whether tranilast administered to pigmented rats inhibits formation of
choroidal neovascularization induced by diode-laser photocoagulation.
Female Brown Norway rats were used. On day 0, choroidal neovascularization was
induced by diode-laser photocoagulation, using a setting of 75 jum spot size, 0.1 second's duration,
and 100 mW intensity. Tranilast (200 or 600 mg/kg per day) was administered orally twice daily for
14 days. Indomethacin (1 and 5 mg/kg per day) was administered orally once a day for 14 days.
Choroidal neovascularization was evaluated on days 7 and 14 by fundus photography and fluorescein angiography. Late-phase fluorescein angiography was scored according to four grades. The
animals were killed on day 14, and the lesions were evaluated histologically.
METHODS.
In the vehicle-treated group, 34 of 35 burns (97%) showed fluorescein staining and late
leakage on day 14. Choroidal neovascularization was identified by light microscopy in all the lesions
that showed fluorescein staining and late leakage. The score of fluorescein staining was reduced in
rats given 200 mg/kg per day or 600 mg/kg per day (P < 0.01) of tranilast. The thickness of the
laser-induced lesions was reduced in a dose-dependent manner by tranilast, a significant difference
was observed with 600 mg/kg per day (P < 0.05). Oral indomethacin treatment did not reduce
fluorescein staining on day 14.
RESULTS.
Tranilast inhibits the development of choroidal neovascularization in this experimental model. (Invest Ophthalmol Vis Sci. 1999;40:459-466)
CONCLUSIONS.
A
ge-related macular degeneration (AMD) is the main
cause of blindness among the elderly in developed
countries.1"3 This condition is subdivided into exudative and dry forms. In exudative AMD, choroidal neovascularization (CNV) extends through Bruch's membrane and into the
subretinal space, subretinal pigment epithelium, or both.6'7
The choroidal neovascular membrane then destroys the macula, which results in visual loss. Although submacular surgery
to remove CNV has been advocated, the efficacy of this new
therapy has yet to be convincingly demonstrated,8"11 and at
present laser photocoagulation is the only well-established
treatment. Unfortunately, however, the outcome of laser photocoagulation is not at all satisfactory. The Macular Photocoagulation Study Group12 reported that laser treatment of subfoveal CNV induced an immediate decrease in visual acuity, and
they also showed that the neovascularization recurrence rate
was rather high.13"15
Recently, antiangiogenic agents such as interferon16 and
thalidomide17 have been used to suppress the development of
neovascular membranes. However, systemic interferon-a has
been shown to produce no real benefit in patients with neo-
From the "Discovery Research Laboratory, R&D, Kissei Pharmaceutical Co., Minamiazumi; the 2 Department of Ophthalmology, Shinshu University School of Medicine, Matsumoto; and the 'Toxicology
Laboratory, R&D, Kissei Pharmaceutical Co., Minamiazumi, Japan.
Submitted for publication January 27, 1998; revised August 7,
1998; accepted September 21, 1998.
Proprietary interest category: P.
Reprint requests: Nagahisa Yoshimura, Department of Ophthalmology, Shinshu University School of Medicine, Matsumoto 390-8621,
Japan.
vascular AMD.18 The Age-Related Macular Degeneration and
Thalidomide Study is evaluating the potential of thalidomide as
an adjunctive treatment for patients with choroidal neovascular membranes associated with AMD. Recently, however, it
was reported that thalidomide did not prevent the recurrence
of a choroidal neovascular membrane in a patient with punctate inner choroidopathy.19
Tranilast is an antiallergic drug whose antiallergic effect is
thought to result from inhibition of the release of chemical
mediators from mast cells and basophils.20'21 Several studies
have also shown that tranilast prevents or reduces the formation of keloids and hypertrophic scars through its inhibition of
abnormal fibroblast proliferation, collagen synthesis, and the
production of cytokines and oxygen-free radicals from activated macrophages and neutrophils.22"24 Furthermore, it has
been reported that tranilast inhibits the proliferation and migration of smooth muscle cells and collagen synthesis by these
cells.25
We have recently shown that tranilast inhibits some steps
in the process of angiogenesis, including chemotaxis, proliferation, and tube formation of microvascular endothelial cells in
vitro, and that it exerts an inhibitory effect on angiogenesis in
vivo.26 In the present study, we examined the effect of the
systemic administration, of-tranilast on experimental CNV created by diode-laser irradiation in pigmented rats.
MATERIALS AND METHODS
Experimental CNV
Female Brown Norway (BN) rats (CLEA Japan, Tokyo, Japan)
weighing between 220 and 300 g were used. All animal exper-
Investigative Ophthalmology & Visual Science, February 1999, Vol. 40, No. 2
Copyright © Association for Research in Vision and Ophthalmology
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Takehana et al.
iments conformed to the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research. Rats were anesthetized with intraperitoneal pentobarbital (50 mg/kg), and the
pupil was dilated by topical phenylephrine and tropicamide.
Diode-laser photocoagulation was carried out using a diodelaser photocoagulator (OcuLight Six; IRIS Medical, Mountain
View, CA) and a slit lamp delivery system with a Nikon slitlamp (model NS-1; Nikon, Tokyo, Japan). A handheld coverslip
served as a contact lens. One or two burns were created
between the major retinal vessels in each eye, using a setting of
75-/Am spot size, 0.1-second duration, and 100-mW intensity, as
described previously.27
Evaluation of Time Course of Experimental CNV
Five BN rats were used to establish the natural time course of
diode-laser-induced CNV. On day 0, laser photocoagulation
was performed as described above. The coagulated lesions
(n = 20) were assessed on days 4, 7, 14, 21, 28, 56, and 301 by
means of ophthalmoscopy, fundus photography, and fluorescein angiography. For fluorescein angiography, 1 ml/kg of 10%
sodium fluorescein (Fluorescite; Alcon, Fort Worth, TX) was
injected into the tail vein of anesthetized rats, and angiograms
were taken using a Kowa fundus camera (GENESIS; Kowa,
Tokyo, Japan).
Evaluation of the Effects of Tranilast and
Indomethacin on Experimental CNV
A total of 96 female BN rats were divided into six groups. All
rats underwent laser photocoagulation as described above.
Group A rats (n = 19) received 100 mg/kg tranilast by mouth
twice daily (total dose, 200 mg/kg per day) for 14 days. Drug
administration was started immediately after photocoagulation
(day 0) and continued until day 13- Group B rats (n = 14) were
given 300 mg/kg tranilast twice daily (total dose, 600 mg/kg
per day) for 14 days using the same schedule as for group A.
Group C (n = 18) rats were used as controls for groups A and
B and received 0.5% carboxymethylcellulose (CMC) by mouth
instead of tranilast twice daily for 14 days. Group D rats (n =
15) received 1 mg/kg indomethacin orally once a day for 14
days. Drug administration was started immediately after photocoagulation (day 0) and continued until day 13. Group E rats
(n = 14) were given 5 mg/kg indomethacin once a day for 14
clays using the same schedule as for group D. Group F (n = 16)
rats were used as the control for groups D and E and received
0.5% CMC instead of indomethacin once a day for 14 days.
Tranilast, vV-(3,4-dimethoxycinnamoyl) anthranilic acid, was
synthesized in our laboratory and suspended in 0.5% CMC.
Indomethacin was purchased from Merck (Rahway, NJ) and
was suspended in 0.5% CMC. The lesions were studied on days
7 and 14 using ophthalmoscopy, fundus photography, and
fluorescein angiography.
Histopathologic Studies
Fourteen days after photocoagulation, the rats were killed
with an overdose of sodium pentobarbital. Eyes were enucleated and fixed overnight in 4% glutaraldehyde in 0.1 M
phosphate buffer, pH 7.4, at 4°C. For light microscopy,
tissue samples were dehydrated and embedded in paraffin.
Then, serial sections were cut at 3 /xm thickness. Each
section was cut perpendicularly to the retina, as confirmed
by the number of nuclei in the inner nuclear layer of the
IOVS, February 1999, Vol. 40, No. 2
healthy retina. Serial sections were stained in an ABC, ABC
sequence with hematoxylin and eosin, Masson's trichrome,
or immunohistochemically with anti-von Willebrand (VW)
factor. Immunohistochemical staining with anti-VW factor
was performed according to the manufacturer's protocol
using the EPOS method (DAKO, Carpinteria, CA). Briefly,
sections were deparaffinized and then treated with 3% hydrogen peroxide to block endogenous peroxidase activity.
After washing with Tris-HCl buffer (pH 7.4), the sections
were incubated with a rabbit anti-human VW factor antibody
(DAKO) for 45 minutes at room temperature, rinsed with
Tris-HCl buffer, and then treated with true blue (Funakoshi,
Kyoto, Japan) for 5 minutes at room temperature. For transmission electron microscopy, eyes were fixed as described
above, postfixed in 1% osmium tetroxide, and dehydrated in
ethanol. After substitution with propylene oxide, the specimens were embedded in Epoxy resin. Thin sections (1 /Ltm)
were stained with 0.05% toluidine blue in 0.1 M phosphate
buffer (pH 7.4). Ultrathin sections were post-stained with
2% uranyl acetate-lead citrate and then examined with a
JEM-1200EX electron microscope OEOL, Tokyo, Japan).
Measurement of the Thickness of Retinal Lesions
A quantitative morphometric assessment of thickness of the
retinal lesions was carried out using a computer-assisted image
analysis system in a masked fashion. Microscopic images
(Olympus BX50 microscope with a working magnification of
X200) of hematoxylin and eosin-stained retinal sections were
acquired via a charge-coupled device video camera (model
MCD-350; Olympus, Tokyo, Japan), digitized by an image-grabber board, and analyzed using analysis software (MacScope,
version 2.3; Mitani, Fukui, Japan). The distance between the
disrupted retinal pigment epithelium and the top of the lesion
was measured. For each retinal lesion, approximately 30 sections were examined, with the thickest value used for the
evaluation. These measurements were made by two examiners
in a masked fashion.
Effects of Tranilast and Indomethacin on
Fluorescein Intensity
Rat serum was prepared from untreated BN rats killed with an
overdose of sodium pentobarbital. One milliliter of serum was
mixed in a glass tube with 10 /xl of 100 mg/ml sodium fluorescein solution and either 5 /xl dimethyl sulfoxide or dimethyl
sulfoxide with various quantities of drugs. After incubation at
room temperature for 5 minutes, the fluorescein intensity was
measured in a fluorescence spectrophotometer (model F-2000;
Hitachi, Tokyo, Japan) with an excitation wavelength of 490
nm and an emission wavelength of 580 nm.
Measurement of Tranilast Concentration in Rat
Plasma
Five Sprague-Dawley rats (SLC, Shizuoka, Japan) were used to
study the pharmacokinetics of tranilast. Blood samples were
collected from each rat at 0.25, 0.5, 1, 2, 3, 4, and 12 hours
after oral administration of 100 mg/kg tranilast. In addition,
three BN rats from group B were killed with an overdose of
sodium pentobarbital after fluorescein angiographic evaluation
on day 14, and blood samples were collected.
All blood samples were centrifuged to provide plasma.
Methanol (2 ml) and internal standard solution (100 jLtg/ml of
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Tranilast Suppresses Experimental CNV
IOVS, February 1999, Vol. 40, No. 2
HOOC
MeO
MeO
FIGURE 1. Chemical structure of tranilast.
AT-cinnamoyl anthninilic acid in acetonitrile, 200 /xl) were
added to the plasma samples (100 /xl) and mixed for 15 minutes. The samples were centrifuged at 3000 rpm for 10 minutes, and the supernatant was subsequently evaporated to
dryness under nitrogen at 40°C. The extract was reconstituted
in 50% acetonitrile (150 ^,1) and acetic acid (10 /u,l), and 50 /txl
was applied to a high-performance liquid chromatography
(HPLC) system. The HPLC system consisted of a liquid chromatograph (model L-4000; Hitachi) with an UV detector, and
recording was carried out at a detection absorbance setting of
350 nm. The HPLC column (Develosil ODS-HG-5 [4.6 X 250
mm]; Nomura Chemical, Seto, Japan) was maintained at 50°C.
The mobile phase consisted of 0.05% trifluoroacetic acid and
acetonitrile (60:40), and it was pumped at a flow rate of 1.0
ml/min.
461
Evaluation and Statistical Analysis
For the evaluation of drug treatment, the intensity of staining in
late-phase (100-140 seconds after dye injection) fluorescein
angiography was graded by two examiners in a masked fashion. Angiograms were graded as follows: Score 0, no staining;
score 1, slightly stained; score 2, moderately stained; score 3,
strongly stained (Fig. 2), and the score was evaluated by a
Wilcoxon signed rank test. When the two scores given for a
particular lesion did not coincide, the higher score was used
for the analysis. Such discrepant scoring was observed in only
12 of the 171 lesions analyzed, and the discrepancy was never
by more than one grade.
The thickness of lesions was evaluated using sections from
the central part of the lesion, which exhibited the thickest
laser-induced retinal destruction. The thickness of the retinal
lesions and the gain in body weight of the rats were evaluated
by ANOVA followed by Dunnett's post hoc test. Values of P <
0.05 were considered statistically significant for all forms of
statistical analysis used. Data are shown as means ± SEM.
RESULTS
Time Course of Changes in Staining Intensity of
Fluorescein Angiograms after Photocoagulation
The time course of changes in the staining intensity in fluorescein angiograms in nontreated rats was evaluated for up to 301
FIGURE 2. Representative late-phase fluorescein angiograms taken 14 days after diode-laser photocoagulation. Photocoagulation was carried out using a setting of 75-fJLm spot size, 0.1-second duration, and
100-mW intensity. One or two burns were created between the major retina! vessels in each eye. The
intensity of fluorescein staining of each lesion in the late-phase angiograms was graded by two independent examiners. The angiograms were graded as follows: score 0, no staining; score 1, slightly stained;
score 2, moderately stained; and score 3, strongly stained.
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IOVS, February 1999, Vol. 40, No. 2
inhibits the intensity of fluorescein staining in experimental
CNV (Fig. 5C).
Thickness of the Lesions
The thickness of the lesions, which was evaluated morphometricaily in serial sections, was 48.7 ± 30 ju.ni (n ~ 33),
43.0 ± 2.9 ^m (n = 21), and 54.9 ± 3.5 /am (n = 32) in groups
A, B, and C, respectively. Thus, lesions were less thick in
tranilast-treated rats, although the effect was statistically significant only at 600 mg/kg per day (P < 0.05; Figs. 4 and 6).
2-
1-
4 7 14 21 28
56
300
Time after photocoagulation (day)
FIGURE 3. Time course of changes in intensity of fluorescein staining
in angiograms taken after diode-laser photocoagulation in nontranilasttreated rats. Each point indicates the mean of the scores of 9 to 20
lesions. Fluorescein staining was first observed 4 days after photocoagutation. Intensity of the staining then increased, and it reached its
peak approximately 14 days after photocoagulation. Strong fluorescein
staining was still present 301 days after treatment, the time of the last
observation.
days after photocoagulation. As shown in Figure 3, weak fluorescein staining was observed on day 4. Thereafter, the intensity increased, reaching its peak on about day 14. Strong fluorescein staining was still present on day 301.
Histopathologic Study
To determine the extent of the extracellular matrix, especially
collagen, in the lesions, we used Masson's trichrome staining.
Fourteen days after photocoagulation, collagen could be seen
in laser-induced lesions by blue staining, and cells could be
identified within the lesions by red staining (graphic data not
shown). In terms of the intensity of collagen staining, there
was no apparent difference between the lesions in the control
and tranilast-treated groups. Electron microscopic evaluation
revealed that new choroidal vessels were present within the
lesions (graphic data not shown), but there was no apparent
difference between the control and tranilast-treated groups.
Body Weight Gain in Rats
There was no significant difference between the control and
tranilast-treated groups in the gain of body weight. The values
were 27.1 ± 3.4g/l4d(n = 13), 19.2 ± 3.1 g/14 d (n = 11),
Choroidal Neovascularization
Because the intensity of fluorescein staining reached its peak
on about day 14, we decided to evaluate the effect of tranilast
on experimental CNY by fluorescein angiography on day 14.
Hyperfluorescence was observed in almost all lesions in group
C animals (control rats for groups A and B). On day 7, nearly all
burns (31/32, 96.9%) showed fluorescein staining and late
leakage; on day 14, 34 of 35 (97.1%) coagulated sites showed
changes suggestive of CNV. Histologic assessment confirmed
the presence of CNV in 100% of those lesions that were graded
as score 1 or higher hyperfluorescence (Fig. 4). Immunostaining of sections with anti-VW factor antibody revealed stained
endothelial cells within the lesion in all burns graded as score
1 or higher (graphic data not shown).
Fluorescein Angiographic Evaluation of CNV
On day 7, group A and group B rats showed a somewhat
weaker staining intensity in fluorescein angiograms than did
group C (control) rats. However, there was no statistically
significant difference between the control group and the tranilast-treated groups (Fig. 5A). On day 14, the score given to
the staining influoresceinangiograms was lower than control
in both tranilast groups, the effect appeared to be dose-dependent (Fig. 5B). In fact, oral tranilast decreased the number of
spots that showed strong dye staining (score 3) and increased
the number showing weak staining (score 1). Group B (600
mg/kg tranilast per day) showed significantly lower scores than
did the control group (P < 0.01).
In contrast, there was no statistically significant difference
between the control and indomethacin-treated groups, although there was noted a tendency that oral indomethacin
^
^
FIGURE 4. light microscopy of retinal lesions from control (A) and
tranilast-treated (B; 600 mg/kg per day) rats 14 days after diode-laser
photocoagulation. Retinal sections were stained with hematoxylin and
eosin. (A) A thin multikiyered fusiform membrane is seen in the central
area of the lesion internal to the choroid. (B) Thin proliferative membranes are seen in a tranilast-treated rat.
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IOVS, February 1999, Vol. 40, No. 2
Tranilast Suppresses Experimental CNV 463
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control
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200
600
tranilast (mg/kg/day)
p<0.01
control
200
600
tranilast (mg/kg/day)
FIGURE 6. Effect of tranilast on thickness of the retinal lesions 14 days
after photocoagulation. Each column shows the mean ± SEM of measurements taken from 21 to 33 sections. Tranilast reduced the thickness of the lesions in a dose-dependent manner. The lesions in the
group treated with tranilast at 600 mg/kg per day were significantly
less thick (P < 0.05) than those in the control group.
B
control
200
600
tranilast (mg/kg/day)
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and 24.0 ± 4.2 g/14 d (n = 12) in groups A, B, and C,
respectively.
Oral administration of 1 mg/kg indomethacin did not affect body weight gain. However, it was decreased in the 5
mg/kg indomethacin-treated group (group E). In group E, 7
animals died during the experiment. The changes in weight
were 132 ± 2.0 g/14 d (n = 15), -0.8 ± 1.2 g/14 d (n = 7),
and 10.2 ± 0.8 g/14 d (n = 16) in groups D, E, and F,
respectively.
Intensity of Fluorescein Staining In Vitro
To confirm that the presence of tranilast or indomethacin does
not influence the grading of the fluorescein angiograms, the
effect of drugs on fluorescein intensity was assessed in vitro. As
shown in Table 1, tranilast had no effect on fluorescein intensity at concentrations of 10 /xg/ml or less. With 100 /xg/ml and
1000 /xg/ml tranilast, the fluorescein intensity was increased to
1.5 and 1.7 times that of control, respectively. Indomethacin
also had no effect onfluoresceinintensity at concentrations of
100 /xg/ml or less.
Concentration of Tranilast in Rat Plasma
control
indomethacin (mg/kg/day)
FIGURE 5. Effect of tranilast and indomethacin on the intensity of
fluorescein staining. Each column shows the mean score; each circle
shows the intensity score offluoresceinstaining in a single lesion. (A)
Effects of tranilast at day 7. There was no statistically significant
difference between the control and tranilast-treated groups. (B) Effects
of tranilast at day 14. The intensity offluoresceinstaining was weaker
As stated above, tranilast affected fluorescein intensity at concentrations of 100 jag/ml or more. For this reason, measurement of the concentration of tranilast in rat plasma after fluorescein angiography was performed on day 14, one day after
in tranilast-treated rats than in control rats. Tranilast at 600 mg/kg per
day showed significantly lower scores than did the control group (P <
0.01). (C) Effects of indomethacin at day 14. There was no statistically
significant difference between the control and indomethacin-treated
groups.
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Takehana et al.
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1. Effect of Tranilast and Indomethacin on
Fluorescein Intensity In Vitro
TABLE
Fluorescein Intensity (%)
Tranilast
0.5% DMSO
0.1 /xg/ml
1 /xg/ml
10 jug/ml
100 /xg/ml
1000 ixe/ml
100
101.3
103.8
108.2
152
171.7
Indomethacin
100
105.2
104.3
102.0
163.2
DMSO, dimethyl sulfoxide.
cessation of treatment with 600 mg/kg per day. The mean
plasma concentration of tranilast was 0.87 /xg/ml (minimum,
0.11 /xg/ml; maximum, 2.26 /xg/ml; n = 3). As can be seen in
Table 1, tranilast at these concentrations did not affect fluorescein intensity.
Pharmacokinetics of Tranilast in Rat Plasma
After oral administration of 100 mg/kg tranilast, the highest
plasma concentration (Cmax), which was 130.9 M-g/ml, was
obtained at 15 minutes, but this concentration then declined
rather quietly; the half-life (t 1/2 ) was 4.5 hours (Fig. 7). Area
under the curve (AUC) was 281.2 /xg-h/ml.
DISCUSSION
Choroidal neovascularization and its sequelae are major causes
of visual loss. Hence, a new therapy for CNV in AMD has been
long-awaited. Animal (monkey) models of laser-induced CNV
were described nearly 20 years ago by Ryan28 and by Archer
and Gardiner.29 Since then many animal models, including
rabbits,30'31 cats,32'33 and rats,34"37 have been developed and
used to evaluate the effect of various antiangiogenic agents on
CNV. Of the numerous animal models, the pigmented rat has
been used by several groups because its retina is similar to the
human retina inasmuch as it is homogeneously pigmented and
exhibits a high incidence of CNV after photocoagulation. Although the CNV induced by laser treatment may differ from
that which occurs naturally in human eyes, this reproducible
model is useful for both basic investigations of choroidal angiogenesis and for studies of its modulation by drug therapy.
In the study described herein, we produced experimental
CNV in the subretinal space of pigmented rats by diode-laser
photocoagulation. Because no detailed study has been published on the time course of CNV after diode-laser photocoagulation in rats, we first studied this aspect of CNV. In our
model, fluorescein-staining associated with CNV was first observed on day 4 (i.e., 4 days after photocoagulation), reached
its peak on day 14, and was still at a high level on day 301 (Fig.
3). On the basis of these findings, we opted to evaluate the
suppressive effect of tranilast on day 14.
On day 14 after diode-laser photocoagulation, nearly all
burns (34/35, 97%) showed fluorescein staining and late leakage suggestive of CNV. In contrast, using krypton laser photocoagulation in pigmented rats, Frank et al.38 reported that CNV
occurred in only 6 of 13 lesions (46%), and Dobi et al.27
reported fluorescein angiographic leakage in only 24 of 86
lesions (28%) after 2 weeks. Wavelength absorption curves
suggest that the transmission of diode-laser energy (780 - 850
nm) through the blood should be equal to or greater than that
of krypton red irradiation.39 Therefore, superior transmission
of diode-laser energy may be one of the reasons why we
succeeded in producing highly reproducible CNV.
In the present study, tranilast administered orally at 200
mg/kg per day and 600 mg/kg per day reduced the intensity of
staining on fluorescein angiograms and the thickness of lesions
in a dose-dependent manner in our experimental model of
CNV (Figs. 5, 6). The dosages of tranilast used in this model are
about the same as the effective dosage used in the experimental allergy,20 keloids and hypertrophic scars,22 and vascular
restenosis studies40 in rats. The present pharmacokinetic data
showed that the maximum plasma concentration was 130.9
/xg/ml after oral administration of tranilast at a dose of 100
mg/kg (Fig. 7). Recently, we reported that tranilast inhibits
several important steps in angiogenesis-namely, proliferation,
vascular endothelial growth factor-induced chemotaxis, and
tube formation by human dermal microvascular endothelial
cells-with IC50 values of 44.5 M-g/ml, 44.2 /xg/ml, and 57.3
ixg/ml, respectively.26 Moreover, at doses of 30 /xg/ml or more
of tranilast inhibited proliferation and collagen synthesis by
fibroblasts derived from human keloids and hypertrophic
scars.23 Because a plasma concentration of more than 30 /xg/ml
was maintained for only 2 hours with a single administration of
100 mg/kg tranilast (Fig. 7), twice daily administration of 100
mg/kg tranilast is the minimal dose in rats to obtain pharmacologically effective plasma concentrations of the drug.
It has been reported that topical treatment with indomethacin suppressed corneal neovascularization via inhibition of
prostaglandin production.41"44 Moreover, intravitreal administration of indomethacin is known to reduce the incidence of
the subretinal neovascularization typically induced by argon
laser photocoagulation in primates.45 However, oral administration of indomethacin did not inhibit CNV in the present
study. It is possible that a sufficient concentration of indomethacin was not obtained in the choroid by the oral administra10001
100"
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o
10
(0
CO
m
a.
2
4
6
8
10
12
14
Time after administration (hr)
FIGURE 7. Plasma concentration of tranilast after a single orally administered dose of 100 mg/kg tranilast in SD rats. Each point represents
the mean ± SEM (n = 5).
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IOVS, February 1999, Vol. 40, No. 2
tion. Higher doses of indomethacin were toxic to the rats
because their weight gain was markedly less than normal, and
half of the animals died during the follow-up period. In contrast
to indomethacin, tranilast does not affect the activity of cyclooxygenase in vitro,46 so the suppressive effects of tranilast on
experimental CNV are not related to the inhibition of cyclooxygenase pathways.
Tranilast has been used clinically in Japan in the treatment
of asthma, allergic rhinitis, allergic conjunctivitis, keloids, and
hypertrophic scars at a dose of 300 mg/d, and it was recently
reported that tranilast reduced the rate of post-percutaneous
transluminal coronary angioplasty restenosis at a dose of 600
mg/d in a phase III clinical trial.47 The doses of tranilast in
clinical use are far less than those used in this rat experiment,
so difference may be explained by different pharmacokinetics
of tranilast between humans and rats. When tranilast (200 mg,
single dose) was administered orally to patients with angina,
the maximum plasma concentration was obtained at 4.7 hours,
and Cmax was 31.7 /Ltg/ml. The t, /2 was 8.4 hours, and the AUC
was 3692 jLtg-h/ml (Hideo Tamai, personal communication,
1997). On the basis of this result, using a nonlinear leastsquares regression computer program (WinNonlin version 1.1;
Scientific Consulting, Cary, NC), we estimated the plasma concentration of tranilast when 200 mg of the drug was administered three times a day for 7 days in humans. The estimated
maximum plasma concentration was 730 ju-g/ml. Using the
pharmacokinetic data obtained in the present study (Cmax =
130.9 jag/ml, time to peak plasma concentration = 0.25 hour,
t I/2 = 4.5 hours, AUC = 281.2 jLtg-h/ml), we also estimated the
plasma concentration of tranilast when the drug was administered twice daily at 100 mg/kg for 7 days in the same manner:
the estimated maximum plasma concentration of tranilast was
142.0 jLtg/ml. Therefore, the maximum plasma concentration
and AUC in humans and rats are almost equivalent.
In our assessment of intensity of fluorescein staining, it is
important to note that the grading system is arbitrary and thus
may not actually reflect a difference in CNV including that
related to AMD. Moreover, tranilast did not extinguish fluorescein staining and leakage, rather it suppressed CNV as assessed
by our grading system. Besides, with tranilast treatment, the
number of eyes that had no leakage at day 14 was not different
from that of the control group. There is a possibility that any
leakage may indicate the lack of efficacy of suppressing laserinduced CNV. However, it is noteworthy that oral indomethacin did not suppress CNV according to the same grading
system.
It is also important to determine whether tranilast affects
the intensity of fluorescein staining. Based on the in vitro
effects of tranilast on fluorescein intensity and on the pharmacokinetic data (Table 1), it is possible that our grading of
photocoagulation spots on day 7 may have been erroneously
high (i.e., we may have underestimated the therapeutic effects
of tranilast at this time point). On the other hand, medication
was stopped the day before the fluorescein angiograms were
obtained on day 14, and on day 14 the concentration of
tranilast in plasma was mvich less than 100 jug/ml. On this
basis, the effect of tranilast on the intensity of staining on the
fluorescein angiograms would have been negligible, which is
true also for indomethacin (Table 1).
Although the data presented in this report show rather
modest effects of tranilast on the inhibition of CNV, to the best
of our knowledge, there are currently no known orally admin-
Tranilast Suppresses Experimental CNV
465
istered drugs that suppress experimental CNV. Even though
the effects of tranilast were modest in the present study, we
believe it is worthwhile to study the clinical application of
tranilast for human AMD, perhaps in combination with more
effective drugs administered locally.
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