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1 From the Department of Pharmaceutical Technology and Biopharmaceutics and the 2 Department of Pharmaceutical Analytical Chemistry, School of Pharmacy, University of Geneva, Switzerland; and the 3 Department of Ophthalmology, Hôtel-Dieu Hospital, Paris, France.
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Abstract |
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METHODS. Trabeculectomy was performed in pigmented rabbit eyes. An ointmentlike formulation of POE containing 1% wt/wt 5-FU was injected subconjunctivally at the site of surgery, during the procedure. Intraocular pressure (IOP), bleb persistence, and ocular inflammatory reaction were monitored until postoperative day 30. Quantitative analysis of 5-FU was performed in the anterior chamber. Histologic analysis was used to assess the appearance of the filtering fistula and the polymer’s biocompatibility.
RESULTS. The decrease in IOP from baseline and the persistence of the filtering bleb were significantly more marked in the 5-FU–treated eyes during postoperative days 9 through 28. Corneal toxicity triggered by 5-FU was significantly lower in the group that received 5-FU in POE compared with a 5-FU tamponade. Histopathologic evaluation showed that POE was well tolerated, and no fibrosis occurred in eyes treated with POE containing 5-FU.
CONCLUSIONS. In this rabbit model of trabeculectomy, the formulation based on POE and containing a precise amount of 5-FU reduced IOP and prolonged bleb persistence in a way similar to the conventional method of a 5-FU tamponade, while significantly reducing 5-FU toxicity.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The antimetabolite 5-Fu is a fluorinated pyrimidine analogue that competitively inhibits thymidylate synthetase, resulting in thymidine deficiency and decreased DNA synthesis. Currently, 5-FU is administered by frequent subconjunctival injections away from the surgical site. Such frequent 5-FU injections are inconvenient for the surgeon and uncomfortable for the patient. Furthermore, therapeutic drug levels are only transiently achieved, necessitating frequent injections and administration of large amounts of drug. Toxic effects on the surrounding ocular tissues are a predictable consequence of this dosage regimen. Toxicity on the conjunctival and corneal epithelium, wound dehiscence, and wound leaks have been reported after repeated subconjunctival administration of 5-FU.3 Topical intraoperative administration of 5-FU with a sponge soaked in a concentrated drug solution has also been used and is associated with adverse side effects, some of which are vision threatening.8 Another disadvantage of this mode of administration is the high drug concentration to which the tissues are exposed during the tamponade as well as the imprecision of the amount of 5-FU delivered to the tissues.
These complications may be reduced by using subconjunctivally implanted drug delivery systems that would provide a localized and sustained release of antiproliferative drugs over an extended period (approximately 2 weeks) and then would disappear, leaving a patent fistula after glaucoma filtering surgery. This 2-week period is critical with respect to inflammatory and fibrotic reactions.9 Drug delivery systems investigated to date include collagen implants,10 11 bioerodible polymers,12 13 14 15 nonbioerodible polymers,16 liposomes,17 and microspheres.18
Poly(ortho esters) (POE) are a new family of hydrophobic, biocompatible,19 20 and bioerodible polymers possessing particularly interesting characteristics. Biodegradability represents a significant advantage over other drug delivery systems, because there is no need to remove the device surgically once all the drug has been released. Kinetics of drug release from POE, as well as polymer degradation rate, are almost constant, without any burst effect, and can be controlled by factors such as polymer molecular weight and the physicochemical properties of the incorporated substances.21 22 The viscous, ointmentlike consistency of the polymeric material allows the incorporation of drugs into the carrier by simple mixing at room temperature, without the use of solvents. POE can be injected using a conventional syringe with an appropriate needle, which is a significant advantage when compared with solid devices that must be placed either with a trocar or through a more complex surgical procedure. Moreover, the viscous properties of POE make its presence within the eye smooth and nontraumatic, because it spreads in the space.
The purpose of this study was to evaluate a sustained drug delivery system based on POE and 5-FU as an adjunct treatment to filtering surgery in rabbits.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The polymer was characterized by infrared spectroscopy, nuclear magnetic resonance and size exclusion chromatography.22 The average molecular weight of the polymer used in this study is 9.3 kDa, with a polydispersity of 1.4. POE is a viscous material, with Newtonian behavior. It is transparent, with a refractive index of 1.47 and is gel-like in appearance.
Preparation of the Formulations
Formulations were prepared under a laminar air-flow hood. The
added drug, 5-FU (Sigma, Buchs, Switzerland), had been 
-sterilized
at 2.0 MRad and homogeneously dispersed in the aseptically prepared
semisolid polymer under aseptic conditions at a concentration of 1%
wt/wt.24
The viscous mixture was conditioned into a 1.0-ml
syringe, each sample being 200 µl (240 mg).
Animals
Pigmented Fauve de Bourgogne female rabbits weighing from 2 to 3
kg, 10 to 12 weeks of age, were used (Jean-Pierre Ravaut;
Institut de la Recherche Agronomique, Nouzilly, France), and
experiments were conducted in accordance with the ARVO Statement for
the Use of Animals in Ophthalmic and Vision Research. Six eyes were
used in each experimental group.
Filtering Surgery
General anesthesia was induced with intramuscular injection of
50 mg/kg ketamine and 15 mg/kg xylazine. With rabbits under local
anesthesia with oxybuprocaine 0.4% (Novesine; Ciba Vision, Basel,
Switzerland), a lid speculum was inserted to expose the globe. A
limbus-based conjunctival flap was reflected. Tenonectomy was performed
to expose the underlying sclera, followed by careful conjunctival
dissection anterior to the limbus. Hemostasis was carefully maintained
with cautery. A half-thickness, limbal-based, 4 x 4-mm scleral
flap was made that extended just anterior to the limbus. At this point,
one group of rabbits received an intraoperative tamponade of 5-FU. A
4 x 1-mm dry section of a Weck-Cel sponge (Edward Weck,
Research Triangle Park, NC) was soaked in a 50-mg/ml 5-FU solution
(Fluorouracil; Teva, Mijdrecht, The Netherlands). The sponge was
placed between the conjunctiva and the sclera over the planned
filtration site for 5 minutes. The corneal epithelium was protected by
applying a wet sponge over the cornea. The treated area was thoroughly
irrigated with 20 ml of balanced salt solution. A 3-mm limbal incision
was made with a 45° blade that entered the anterior chamber. A block
of tissue containing inner sclera, trabeculum and peripheral cornea,
measuring approximately 3 x 1 mm, was excised at the limbus. A
peripheral iridectomy was then performed. The scleral flap was
approximated with two 10-0 nylon sutures. The conjunctiva was
repositioned and the wound closed with 8-0 Vicryl suture (Ethicon,
Piscataway, NJ) in a continuous fashion. Just before the last step, a
0.9-mm needle (20-gauge) was inserted in the subconjunctival space, and
200 µl of polymer mixture was injected adjacent to the
trabeculectomy. The suture was closed tightly, and topical neomycin
ophthalmic ointment was applied.
Four different groups were tested (n = 6): group I, eyes that underwent trabeculectomy alone; group II, eyes that received 5-FU as an intraoperative tamponade; group III, eyes that received POE alone; and group IV, eyes that received POE containing 1% wt/wt 5-FU.
Clinical Observations
Slit-lamp observations were performed approximately every 2 days
for 30 days after surgery to assess the filtering bleb status and the
overall inflammatory state of the eye. Intraocular pressure (IOP) was
measured at the same time intervals using Goldmann applanation
tonometry, with rabbits under local anesthesia, and was compared with
the preoperative IOP.
Statistical analysis was performed to compare experimental data with data from control eyes, using a nonparametric Mann–Whitney test. The following variables were analyzed: IOP, time to bleb failure, conjunctival hyperemia, and corneal edema. P < 0.05 was considered significant.
Quantitative Determination of 5-FU
Concentrations of 5-FU were determined by liquid chromatography
(LC) using a high-performance liquid chromatography (HPLC) system (LC
module I plus; Waters, Milford, MA), consisting of a power line
controller pump (model 600E) an autoinjector (715
Ultra WISPTM), a tunable absorbance UV detector set at 268 nm, and a
software integrator (Millennium) all from Waters. Separation was
performed with a C18 HPLC column (Nucleosil 100-5; Macherey Nagel,
Düren, Germany; 250 x 4.0 mm; internal diameter, 5 µm).
The mobile phase consisted of ammonium phosphate buffer (50 mM; pH 6.0)
delivered at 1.0 ml/min. Before use, the mobile
phase was filtered through a 0.45-µm membrane filter (Millipore,
Molsheim, France) and degassed 10 minutes by
sonication.
At postoperative days 1, 2, 5, 7, and 14, a paracentesis was made with rabbits under local anesthesia, and 100 µl of aqueous humor was collected for quantitative analysis. Samples and quantitative standards were homogenized and directly injected into the HPLC system, with an injection volume of 20 µl.
Histologic Analysis
Rabbits were killed at different time points ranging from 5 to
30 days after surgery by intracardiac injection of a lethal dose of
pentobarbital, and their eyes were enucleated and fixed to be studied
histologically by conventional optical microscopy.
Anteroposterior sections were stained with hematoxylin-eosin to examine
the conjunctiva, the iridocorneal angle, and the appearance of the site
of the bleb. Every sample was treated simultaneously to reduce
variations among fixation procedures.
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Results |
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At the site of surgery, trabeculectomy also triggered reversible edema of the cornea, which reached its apex at day 5. The presence of POE triggered edema of the same intensity, which was also reversible. In contrast, 5-FU applied during surgery as a tamponade triggered more severe edema, extending to half the cornea (Fig. 2 II) . The frequency and the severity of corneal edema in the 5-FU tamponade group was significantly higher than in any other group. Superficial punctuate keratitis occurred in two eyes. The group that received POE+5-FU also showed some edema, but it remained localized at the surgical site and was not significantly different from that in the control groups (Fig. 2 IV) .
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Bleb Appearance and Survival Curves.
In group I (control eyes that underwent trabeculectomy alone), all
blebs were elevated and typically had sharply demarcated edges a few
days after surgery. Fifty percent of blebs had collapsed by 9 days and
100% by 19 days. In group II (eyes that received an intraoperative
5-FU tamponade), the blebs were significantly more persistent, lasting
from day 12 until the end of the experiment (day 29). Group III, which
received POE alone, showed delayed persistence of the bleb compared
with group I (Fig. 2 Iand III)
, but eventually all blebs failed. All
blebs in group IV (POE containing 5-FU) survived until postoperative
day 19, and 83% survived after 1 month. There were no significant
differences between group IV and group II. Bleb survival curves for the
four groups are displayed in Figure 3
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In all eyes that received POE+5-FU, quantitative determination of 5-FU in the aqueous humor showed detectable amounts of 5-FU (below 0.5 µg/ml) for 2 weeks, which shows that POE was still present in the subconjunctival space for that period and released 5-FU in a continuous fashion. In particular, no burst release of 5-FU was observed in the early postoperative period, according to observations for in vitro 5-FU release.22 Thus, at each time point, a minimal amount of 5-FU was present in the anterior chamber, corresponding at least to 3 orders of magnitude below the threshold concentration for 5-FU toxicity to the corneal endothelium (1–10 mg/ml) reported by Mannis et al.25
5-Fluorouracil was not quantified in the anterior chamber of eyes that received an intraoperative tamponade for several reasons: 5-FU is applied and rinsed off before opening the anterior chamber, and therefore no 5-FU should penetrate into the eye; 5-FU soaks the surrounding tissues and does not diffuse into the anterior chamber; moreover the flux direction is opposite to the direction of the possible 5-FU intracameral penetration; and last, in all the published literature concerning 5-FU intraoperative tamponade,4 6 8 26 27 none of the authors ever measured the 5-FU concentrations in the anterior chamber.
Histology
In control animals killed at 5 days after surgery, a bleb was
present, and the trabeculectomy was patent. Some inflammatory cell
(polymorphonuclear cells) infiltration was observed around the margins
of the scleral flap. The edges of the iris around the iridectomy had
partially prolapsed into the fistula in some specimens, although they
did not completely block it. No evidence of any conjunctival filtration
could be seen.
Bleb failure occurred at 10 days in eyes that underwent trabeculectomy alone (group I). Eyes showed evidence of inflammatory cell infiltration and fibrovascular tissue at the surgical site. The trabeculectomy and bleb closed due to bulk filling by granulation tissue and fibroblasts.
Until the end of the experiment, eyes that received 5-FU, either as a tamponade or incorporated in POE, showed no signs of fibrosis of the filtration fistula. The scleral flap margins were absent from any fibrotic subconjunctival fibroblasts (Fig. 6) . The conjunctival filtration was efficient, visible as a small cavity surrounded by fibrin and cells. Some slight modifications of the iris were observed, notably pigment redistribution. These modifications of the uvea were more marked in the 5-FU tamponade group in which significant pigment alterations and pigment migration into the external tunica were observed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intraoperative 5-FU had a marked effect on IOP that lasted longer than is reported in the literature. Khaw et al.26 reported a significant difference from the control from day 5 to day 18 for the same dosage regimen in rabbits. In our model, filtering blebs lasted at least 1 month. However, intraoperative application of 5-FU triggered severe complications, such as corneal edema and epithelial defects.
The administration of POE containing 5-FU promoted the success of glaucoma filtering surgery in rabbit eyes. Eyes that received POE and 5-FU after trabeculectomy had lower IOP for at least 1 month, and they showed filtration blebs and patent fistulas, as supported by the histopathologic findings. This improvement in the duration of lowered IOP in the experimental eyes was due to the slow and continuous release of 5-FU from the polymeric matrix, inducing facilitated outflow. In both groups that received 5-FU, no abnormal morphologic changes were seen in the ciliary body epithelium and trabecular meshwork when compared by light microscopy with control eyes. When incorporated in POE, 5-FU also appeared to trigger less toxic reactions, such as corneal edema than when administered during surgery.27 The slow release of 5-FU from POE prevented the focal attainment of high and toxic drug levels. Clinical observations showed that eyes that received POE with 5-FU had reactions similar to those in control eyes. There was some conjunctival hyperemia and corneal edema, but the intensity was comparable to that in control eyes.
POE is a new biomaterial with potential as a controlled release system for drugs as an adjunct treatment in glaucoma filtering surgery. Sustained release of an antiproliferative drug such as 5-FU from a POE carrier means that only one subconjunctival injection must be performed at the time of surgery and enables a precise and small amount of drug to be administered. In contrast, during an intraoperative tamponade, tissues are exposed to a high concentration of antiproliferative drug that is potentially toxic to the corneal epithelium,28 and they are soaked with a very variable, uncontrolled, and unpredictable amount of drug that diffuses rapidly from the sponge. Moreover, the 5-FU solution is basic (pH 9.0) to increase drug solubility, which can also trigger a more acute inflammatory response. We showed that both techniques—that is, the incorporation of a low amount of 5-FU (2.4 mg) in POE or an intraoperative tamponade with a 50-mg/ml 5-FU solution—showed comparable results in IOP and bleb persistence. The incorporation of 5-FU in POE also reduced the toxicity of the drug, because lower amounts had to be used.
Although the results described in previous studies with other biomaterials are very encouraging,13 our system provides several additional intrinsic advantages. Regarding physical properties, the viscous consistency of POE makes it slightly traumatic to ocular structures, compared with solid devices.13 After injection, POE spreads under the conjunctiva and assumes the curve of the ocular globe. Concerning chemical characteristics, the hydrophobic backbone of the polymer allows surface erosion and thus a linear and almost constant release of drugs.22 Burst release, which is often encountered with poly(DL lactic-co-glycolic acid) PLGA-based systems,29 is avoided. The burst release of a drug such as 5-FU with its associated toxicity could have dramatic effects on tissue tolerance. Also, POE has shown excellent biocompatibility in several parts of the eye,20 notably under the conjunctiva.19 Compared with reported results with collagen11 or PLGA,30 POE is better tolerated. Another advantage is that POE does not become encapsulated, because it is progressively resorbed in approximately 2 weeks, in contrast to PLGA-based systems, which degrade more slowly than the drug is released and thus present a risk of encapsulation.13
All animal models of glaucoma filtering surgery have advantages and disadvantages. This study demonstrates a consistent and predictable response of the tissues in the normal rabbit, with the trabeculectomy closing and the bleb collapsing within a short and convenient time period. The overall pattern of wound healing is similar to scars found in humans,31 although more rapid. It has also been reported32 that a significant reduction in IOP may occur, even in the absence of a patent trabeculectomy, and bleb function is thus a more reliable index to assess the fistula’s efficacy. Also, there are numerous anatomic differences between rabbit and human eyes. Notably, eyeball motion is more frequent in man, eyelid pressure is higher, and the iris is less prone to incarcerate the channel in rabbits than in man, so the potential for the mixture to ooze into the anterior chamber is higher in patients. However, we have seen that the concentrations obtained in the rabbit anterior chamber after the administration of the POE 5-FU formulation were 103 times lower than reported toxic concentrations. As a consequence, it is improbable that 5-FU concentrations in the human anterior chamber would increase 3 orders of magnitude; however, this should be investigated.
This newly developed drug delivery system based on bioerodible POE has potential for clinical applications in glaucoma filtering surgery in patients who are at high risk of failed surgery. Scarring at the filtration site can be inhibited by a localized and sustained release of a very small yet efficient amount of the antifibroblastic agent 5-FU, therefore avoiding the need for frequent subconjunctival injections and decreasing toxic ocular side effects caused by intraoperative topical administration of higher amounts of drug. POE combines the advantage of a hydrophobic polymer, allowing a slow release of the hydrophilic drug 5-FU for 2 weeks, with a relatively fast biodegradability. Its proven biocompatibility makes it a biomaterial of choice to deliver drugs in a sustained, controllable way. A study over 6 months is currently under way to assess a long-term follow-up.
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Acknowledgements |
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Footnotes |
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Submitted for publication June 9, 2000; revised November 20, 2000; accepted December 5, 2000.
Commercial relationships policy: N.
Corresponding author: Robert Gurny, Department of Pharmaceutical Technology and Biopharmaceutics, School of Pharmacy, University of Geneva, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland. robert.gurny{at}pharm.unige.ch
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), group II
(
), group III
(
), and group IV
(
). n = 6, mean ± SD. *Significant difference
from group I (P < 0.05).
