Download Iontophoresis of 5-fluorouracil into the conjunctiva and sclera.

No. 3
Reports
583
Investigative Ophthalmology & Visual Science, Vol. 30, No. 3, March 1989
Copyright © Association for Research in Vision and Ophthalmology
Iontophoresis of 5-Fluorourocil into the Conjunctiva ond Sclero.
Mosoki Kondo ond Makoto Aroie
It was investigated in the rabbit eye whether the method of
ionotophoresis could introduce a sufficient amount of 5-fluorouracil (S-FU) to inhibit fibroblast proliferation into the
conjunctiva and sclera, thus considerably reducing the total
amount of 5-FU which must be given to the eye. Five-FU
was introduced with a simple apparatus which was filled
with 5% 5-FU solution and connected to the negative pole of
a current source. When the apparatus was placed over the
cornea, the 5-FU penetration into the cornea showed a correlation with strength of current (0-0.75 mA). When the
apparatus was placed over the bulbar conjunctiva, a sufficient amount of 5-FU to inhibit fibroblast proliferation was
introduced into the conjunctiva and sclera with a current of
0.5 mA passed for 30 seconds. Immediately after iontophoresis, mean 5-FU concentrations in the conjunctiva and
sclera at the iontophoresis site were 480 and 168 iig/g,
respectively. They decreased to 0.6 and 1.2 jig/g by 10 hr,
but were still above the reported IDS0 levels for the cultured
conjunctiva! fibroblast. On the other hand, the total amount
of 5-FU introduced into the eye averaged only 3.7 /Kg and
the current used was much lower than that formerly applied
to the patient's cornea. Invest Ophthalmol Vis Sci 30:583585,1989
Postoperative subconjunctival 5-fluorouracil
(5-FU) injections have been reported to increase the
success rate of filtering surgery with poor surgical
prognosis without serious complications.1 Usually, 5
mg of 5-FU is injected subconjunctivally on the side
opposite to the surgical site1 and the injected 5-FU is
thought to reach the filtering site by diffusing in the
subconjunctival space or via the anterior chamber.
Meanwhile, most of the 5-FU injected is thought to
be absorbed in the surrounding tissues without
reaching the filtering site, causing potentially toxic
effects.2 Furthermore, subconjunctival injections
carry potential risks of bleeding, infection, scarring
and ocular penetration.
Iontophoresis, an old technique in ophthalmology
for introducing drugs into the eye, can efficiently introduce high levels of ionized drugs in a relatively
limited area of the ocular surface without making a
needle hole in the conjunctiva.3 Since the pKa of the
5-FU is 8.04 and the pH of the commercially available 5-FU solution is 8.4, 70% of 5-FU is ionized and
negatively charged in the solution.4 The purpose of
the current study is to determine if the method of
iontophoresis can introduce a sufficient amount of
5-FU into the conjunctiva and sclera to considerably
reduce the total amount of 5-FU which must be given
to the eye to inhibit the fibroblast proliferation.
Materials and Methods. Animals and drug: Japanese albino rabbits of both sexes weighing 2.5-3.0 kg
were used. A solution of 5.0% 5-FU for intravenous
injection (Kyowa Hakko Kogyo Co., Ltd., Tokyo,
Japan) was used throughout the study. The solution
had a pH of 8.4 and osmolality of 865 mOsm, and
contained 5.0% 5-FU and 8.47% tris(hydroxymethyl)aminomethane (NH2C(CH2OH)3), which was added
to enhance the solubility of 5-FU, in distilled water.
Iontophoresis experiment: Before iontophoresis, a
rabbit was anesthetized with 1 g/kg of intraperitoneal
urethane and one drop of 0.4% oxybuprocaine hydrochloride was instilled in the eye. A simple iontophoresis apparatus (Fig. 1) wasfilledwith 5-FU solution; a
neutral electrode, smeared with electrode paste, was
taped to the rabbit's ear.
As the first series of the experiment, the apparatus
connected to the negative pole of a constant current
source was applied to the central part of the cornea
for 30 seconds with a current passed of 0,0.25,0.5 or
0.75 mA. Immediately thereafter, the eye was rinsed
with physiological saline and 2 min later, the rabbit
was sacrificed by an overdose of sodium pentobarbital and the whole cornea excised, blotted and
weighed. The concentration of 5-FU in the cornea
was determined as described below.
As the second series of the experiment, the eye was
proptosed, and the apparatus placed onto the bulbar
conjunctiva 4 mm posterior to the limbus in the superior temporal quadrant. A current of 0.5 mA was
passed for 30 seconds and the eye rinsed with physiological saline. The rabbit was sacrificed immediately
thereafter, or at 0.5, 1, 2, 3, 5 or 10 hr after iontophoresis, and a piece of bulbar conjunctiva (about 8
X 8 mm) at the iontophoresis site was excised, blotted
and weighed. After enucleation, the eye was frozen
with dry ice-acetone mixture. A piece of the anterior
sclera (about 8 X 8 mm) at the iontophoresis site was
excised and weighed. In rabbits sacrificed at 0.5, 1,2
and 3 hr, about 100 yul of the aqueous was obtained by
paracenthesis before freezing. In those sacrificed at
0.5 and 1 hr, the iontophoresis site was examined by
means of a portable slit-lamp microscope immediately after finishing the iontophoresis.
The sample was stored at -80°C until measured.
After thawing, each sample was mixed with pre-
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933147/ on 06/18/2017
584
Vol. 30
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / March 1989
Table 1. Effect of varying the current on 5-FU
transport into the cornea
5% 5-FU
solution
Current
Concentration
0 mA
0.25 mA
0.50 mA
0.75 mA
1.8 ±0.2
5.8 ± 1.5
17.3 ±2.8
32.0 ± 2.2
Duration of iontophoresis is 30 seconds. Values are given as mean and
SEM in four samples.
and 165 /«g/g, respectively. They decreased to 0.6 and
1.2 /tg/g by 10 hr. The 5-FU concentrations in the
aqueous were much lower than those in the conjunctiva and sclera (Table 2, Fig. 2). The total amount of
5-FU introduced was estimated by multiplying the
weights of the conjunctiva and sclera samples obtained just after iontophoresis by the 5-FU concentrations measured in them, and averaged 3.7 ± 0.7
(SEM) /tg (n = 6).
Biomicroscopic observations just after iontophoresis showed no apparent abnormalities in the conjunctiva where the apparatus made contact. After fluorescein staining, however, an irregular-shaped surface erosion of the conjunctiva was seen at that place
in all eyes examined.
Discussion. Since the activity measured by microbiological assay represents 5-FU and its active metabolites, but not its inactive metabolites or degradation
products,7 the 5-FU levels determined by this method
reflect the biological activity of this antiproliferative
drug better than levels determined by using radioactive materials.
The concentration for 50% inhibition of cultured
rabbit conjunctival fibroblast proliferation (ID50) was
reported to be 0.2-0.5 jtg/g and that for cultured
human dermal fibroblast, 0.35 ng/g.8"10 Since the failure of filtering surgery is thought to be due to the
postoperative scarring process in which fibroblasts
play an important role," the subconjunctival or intrascleral space at the filtering site is the area where a
therapeutic level of 5-FU concentrations must be
achieved. After iontophoresis under the conditions of
this study, the 5-FU levels in the conjunctiva and
semipermeable membrane
(molecular cut 25.000)
Fig. 1. Apparatus used for iontophoresis of 5-FU. Semipermeable membrane is a cellulose dialysis membrane with a molecular
cut-off of 25,000 daltons.
weighed sterile 0.1 M phosphate buffer, and the cornea, conjunctiva and sclera samples were homogenized separately and centrifuged at 1560 g for 10 min.
The concentration of 5-FU in the supernatant was
assayed by a standard agar-diffusion bioassay using
Mueller Hinton broth seeded with Micrococcusflavus
ATCC 10240 as the test organism.5-6 To serve as a
blank, the samples of the aqueous, cornea, conjunctiva and sclera were obtained from untreated rabbits,
frozen, stored and processed in the same manner.
Standard solutions were prepared by diluting the
5-FU solution with sterile 0.1 M phosphate buffer.
The 5-FU level in a tissue sample was calculated by
dividing the total amount of 5-FU in the supernatant
by the weight of the sample. The sensitivity of the
bioassay method used was 0.003 >tg/ml.
All the investigations herein conform to the ARVO
Resolution on the Use of Animals in Research.
Results. The mean 5-FU concentration in the cornea increased as the passed current increased (Table
1). Immediately after iontophoresis onto the bulbar
conjunctiva with a current of 0.5 mA passed for 30
seconds, the 5-FU concentrations in the conjunctiva
and the sclera at the iontophoresis site averaged 480
Table 2. Time change of 5-FU concentration after iontophoresis
Hours after iontophoresis
Conjunctiva, jig/g
Sclera, ng/g
Aqueous, *ig/g
0
0.5
1
2
3
5
10
480 ± 77
165 ± 25
50 ±24
21 ± 8
1.1 ± 0.3
6.3 ±1.9
4.4 ± 1.7
0.4 ±0.1
1.4 ±0.5
0.9 ±0.2
0.3 ±0.0
0.9 ± 0.2
1.5 ±0.5
0.1 ±0.0
1.4 + 0.4
1.5 ±0.3
0.6 ± 0.2
1.2 ±0.4
The current and duration of iontophoresis are 0.5 mA and 30 seconds, respectively. Values are given as mean ± SEM in four to six samples.
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933147/ on 06/18/2017
585
Reports
No. 3
According to Duke-Elder,3 the current applied to
the patient's cornea by iontophoresis should not exceed 1-3 raA for a period of 2 min. We used a current
of 0.5 mA passed for 30 seconds, which is much lower
than the above limit. However, surface erosion of the
conjunctiva where the apparatus made contact probably indicates damage caused by the current passed.
The effect of this surface erosion on the integrity of
the nitration bleb would be first studied before applying this method to postoperative eyes.
Iontophoresis affords a simple and effective way to
introduce 5-FU into the conjunctiva and sclera. Besides application to postoperative glaucomatous eyes,
this method may be potentially useful in reducing the
recurrence of pterygia or localized malignancies of
the corneal or conjunctival surface.
9
10
HOURS AFTER IONTOPHORESIS
Fig. 2. Concentration changes of 5-FU in the conjunctiva (•)
and sclera (O) at the iontophoresis site. Each point and bar indicate
mean ± SEM.
sclera were higher than the above ID 5 0 level for at
least 10 hr.
The 5-FU levels in these tissues remained almost
unchanged from 2 to 10 hr, and this may indicate
that this drug binds with some of the tissue components. If so, the 5-FU levels as measured by the
present method would rather reflect the diffusable
5-FU levels and the total amount of 5-FU in the tissue may be somewhat higher than the value here obtained. However, the diffusable 5-FU is thought to be
that which is taken into the fibroblasts in the tissue
and therefore biologically important.
The amount of 5-FU introduced by iontophoresis
in our experiments was only about 4 tig, which was
less than 0.1 % of the dose usually given to patients by
subconjunctival injection.1 Accordingly, the 5-FU
level in the aqueous after iontophoresis in this study
was also much lower than the levels after subconjunctival injection.12 The most frequently observed
complications of postoperative 5-FU subconjunctival
injections are corneal epithelial defects and conjunctival wound leaks,1 which are attributable to the drug
effects on the normal tissues outside of the nitration
site. Thus, the smaller the total amount of 5-FU
given, the fewer the side effects. At pH 8.4,60% of the
tris(hydroxymethyl)aminomethane, which is added
to the solution, remains undissociated, while 40% of
it is ionized and positively charged (data offered by
Kyowa Hakko Kogyo Co., Ltd.). This implies that
negatively charged 5-FU is more selectively introduced into the eye by iontophoresis than by subconjunctival injection or instillation.
Key words: 5-fluorouracil, iontophoresis, conjunctiva,
sclera, rabbit
From the Department of Ophthalmology, University of Tokyo
School of Medicine, Tokyo, Japan. Submitted for publication: May
9. 1988; accepted September 30, 1988. Reprint requests: Makoto
Araie, MD, Department of Ophthalmology, University of Tokyo
School of Medicine, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113, Japan.
References
1. Rockwood EJ, Parrish RK II, Heuer DK, Skuta GL, Hodapp
E, Palmberg PF, Gressel MG, and Feuer W: Gaucoma filtering
surgery with 5-fluorouracil. Ophthalmology 94:1071, 1987.
2. Knapp A, Heuer DK, Stern GA, and Driebe WT Jr: Serious
corneal complications of glaucomafilteringsurgery with postoperative 5-fluorouracil. Am J Ophthalmol 103:183, 1987.
3. Duke-Elder S: The method of administration of drugs. In System of Ophthalmology, Vol. VII, Duke-Elder S, editor. London, Henry Kimpton, 1962, pp. 485-520.
4. Newton DW and Kluza RB: pKa values of medical compounds
in pharmacy practice. Drug Intelligence and Clinical Pharmacy 12:546, 1978.
5. Clarkson B, O'Connor A, Winston L, and Hutchison D: The
physiologic disposition of 5-fluorouracil and 5-fluoro-2'-deoxyuridine in man. Clin Pharmacol Therap 5:581, 1964.
6. Fujita H, Ogawa K, Nakagawa H, Kawaguchi K, Nakagawa Y,
and Doi Y: Pharmacokinetics of 5'-deoxy-5-fluorouridine (5DFUR) by oral administration. J Jpn Soc Cancer Therap
18:916, 1983.
7. Myers CE: The pharmacology of thefluoropyrimidines.Pharmacol Rev 33:1, 1981.
8. Blumenkranz MS, Claflin A, and Hajek AS: Selection of therapeutic agents for intraocular proliferative disease. Arch Ophthalmol 102:598, 1984.
9. Mallick KS, Hajek AS, and Parrish RK II: Fluorouracil (5-FU)
and cytarabine (Ara-C) inhibition of corneal epithelial cell and
conjunctival fibroblast proliferation. Arch Ophthalmol
103:1398, 1985.
10. Blumenkranz MS, Hartzer MD, and Hajek AS: Selection of
therapeutic agents for intraocular proliferative disease: II. Differing antiproliferative activity of the fluoropyrimidines. Arch
Ophthalmol 105:396, 1987.
11. Skuta GL and Parrish RK II: Wound healing in glaucoma
filtering surgery. Surv Ophthalmol 32:149, 1987.
12. Rootman J, Tisdall J, Gukauskas G, and Ostry A: Intraocular
penetration of subconjunctivally administered l4C-fluorouracil in rabbits. Arch Ophthalmol 97:2375, 1979.
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933147/ on 06/18/2017