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1 From the Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison; the 2 Department of Ophthalmology, Mayo Clinic, Rochester, Minnesota; and the 3 Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.
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Abstract |
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METHODS. Topical LAT-A or LAT-B was administered to one eye, and vehicle to the other. IOP was measured by Goldmann tonometry, AHF and corneal endothelium transfer coefficient (ka) by fluorophotometry, [protein]AC by Lowry assay, corneal endothelial cell morphology by specular microphotography, and corneal thickness by ultrasound pachymetry.
RESULTS. LAT-A began to lower IOP at 6 hours and maximally reduced IOP by 4.6 mm Hg at 9 hours. LAT-B lowered IOP within 1 hour and maximally reduced IOP by 3.1 mm Hg at 6 hours. LAT-A increased AHF by 87% for 3 hours and increased ka by 94% over 6 hours; LAT-B increased ka by 39% over 6 hours without affecting AHF. LAT-A increased IV fluorescein entry into the cornea approximately 10 fold, but did not affect IV fluorescein entry into the AC. LAT-A increased [protein]AC by 25% at 2 hours but not 5.5 hours. LAT-B variably and insignificantly increased [protein]AC at 1 hour but not at 6.5 hours. LAT-A induced extensive corneal endothelial pseudoguttata within 1 hour, with normal cell counts by 7 days. LAT-B increased central corneal thickness maximally by 47 µm at 3.5 hours.
CONCLUSIONS. LAT-A and -B significantly reduced IOP and were consistent in their facility-increasing effect, indicating that pharmacologic disorganization of the actin cytoskeleton in the trabecular meshwork by latrunculins may be a useful antiglaucoma strategy. However, effects on corneal endothelium or ciliary epithelium are a potential safety issue.
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Introduction |
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Although latrunculins increase outflow facility and may thus have potential for anti-glaucoma treatment, it is not yet clear whether they reduce introcular pressure (IOP) or affect other ocular tissues, such as the cornea and ciliary body, which is important for safety considerations. Additionally, given their contrasting potency profiles in cultured cells versus the live monkey eye,4 6 8 it would be interesting to know whether LAT-A and LAT-B act differently on IOP or other ocular tissues, perhaps leading to identification of related agents that have a potent effect on IOP but less effect on the ciliary body or cornea. We therefore studied the effects of LAT-A and LAT-B on IOP, aqueous humor flow (AHF), anterior segment fluid barrier and permeability characteristics, and corneal endothelial morphology and function in living monkey eyes.
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Methods |
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Animals and Anesthesia
Adult cynomolgus monkeys (Macaca fascicularis) of both
sexes, weighing 2.0 to 5.5 kg, were studied. Anesthesia was induced by
intramuscular (IM) ketamine (10 mg/kg) and maintained with supplemental
injections of ketamine as required (5 mg/kg every 30 to 45 minutes).
Between ketamine injections during a given experiment, monkeys were
maintained in transfer cages after each measurement and usually were
waking up. The duration of each experiment was 9 hours or less. Some
animals received an extra single IOP measurement at 24 hours after a
9-hour experiment; they were allowed to fully recover from the
anesthesia in their regular cages in the Animal Care Unit between the
9- and 24-hour measurements. Those monkeys used in the specular
microscopy protocol also received IM acepromazine (1 mg/kg). All
experiments were conducted in accordance with the ARVO Statement for
the Use of Animals in Ophthalmic and Vision Research and in compliance
with National Institutes of Health and University of Wisconsin
guidelines.
Slit Lamp Biomicroscopy
A trained ophthalmologist examined all eyes by slit lamp for
integrity of the corneal epithelium and endothelium, presence of flare
or cells in the AC, and clarity of the lens. All animals were free of
ocular abnormalities when studied.
Intraocular Pressure
IOP was determined with a minified Goldmann applanation
tonometer,11
using a cream–milk combination (Half and
Half; Borden, Columbus, OH) as the tear film indicator,12
with the monkey lying prone in a head holder and the eyes positioned 4
to 8 cm above the heart. All monkeys were examined by slit lamp before
the first IOP measurement in each protocol. For each eye, two or three
IOP measurements were averaged as a baseline.
After baseline IOP was measured, 21 or 42 µg LAT-A or 4 µg LAT-B was administered to the central cornea of supine monkeys in one eye and the vehicle to the opposite eye. Blinking was prevented with lid speculums during and for 5 minutes after drug administration (taking care to avoid touching the globe), to maximize drug penetration into the AC and minimize systemic absorption. After drug administration, the speculums were gently removed, and the monkeys were kept supine for another 15 minutes to further facilitate penetration of drug–vehicle solution into the AC. For LAT-A, IOP was then measured every hour for 7 to 9 hours, beginning 1, 6, or 15 hours after the 21-µg dose or beginning 1 or 6 hours after the 42-µg dose, and again at 24 hours. Each protocol included two groups of monkeys. Group one (six monkeys) underwent IOP measurement from 1 to 9 hours and again at 24 hours after the drug; group two subjects (six monkeys) were measured from 6 to 13 hours for both doses and again from 15 to 21 hours on a separate occasion for the 21-µg dose. For both doses, one monkey in group one was used again in group two; therefore, this subject’s two readings, obtained at baseline or the period 6 to 9 hours after the drug on the two different occasions were averaged for data analysis. Thus, n for baseline and the period 6 to 9 hours after drug administration is 11 rather than 12. During intervals between the postdrug reading at 9 hours and that at 24 hours for group one or between the drug administration and the first postdrug reading at 6 hours for group two, the animals were returned to the Animal Care Unit for recovery. For LAT-B, IOP was measured every hour for 6 hours, beginning 1 hour after the 4-µg dose, and again at 24 hours. The animals were returned to the Animal Care Unit for recovery between the measurements at 6 and 24 hours. Anterior segments were examined by slit lamp at 3, 6, 10, or 24 hours after LAT-A, or at 1, 3, 6, or 24 hours after LAT-B.
Aqueous Humor Flow and Corneal Endothelial Permeability
AHF rate was determined noninvasively by scanning ocular
fluorophotometry (Fluorotron Master; Coherent, Palo Alto, CA, and
Ocumetrics, Mountain View, CA). Each monkey was examined
biomicroscopically, and background fluorescence in the cornea and AC
was determined before fluorescein administration. On the afternoon
preceding LAT-A or B administration (usually ~4–5 PM), one drop of
0.5% proparacaine HCl (Alcaine, Alcon) was administered bilaterally
(to enhance corneal penetration of fluorescein) to supine
ketamine-anesthetized monkeys. Five minutes later, five 2-µl drops of
2% fluorescein Na (Alcon) were applied to the central cornea
bilaterally at 30- to 60-second intervals. Blinking was prevented
between drops and for 5 minutes after the final drop with lid
speculums. The next morning (usually ~9–10 AM), after a saline flush
of the conjunctival sac, four 5-µl drops of 5 mM LAT-A (42 µg) or
four 5-µl drops of 500 µM LAT-B (4 µg) were administered to the
central cornea of one eye, and four 5-µl drops of vehicle (25% DMSO)
to the opposite eye, with 1-minute intervals between drops in each eye
with the monkey supine. Blinking was prevented as described. Beginning
30 minutes later, corneal and AC fluorescence was measured every 30
minutes for 6 hours. AC volume was estimated from corneal thickness, AC
depth, corneal curvature, and corneal diameter, all determined
optically.13
AHF rate and
ka (the transfer coefficient for
fluorescein exchange across the corneal endothelium into the AC,
calculated as the area of the corneal endothelium divided by the volume
of the cornea and multiplied by the permeability coefficient of
endothelium from aqueous to cornea14
) were then calculated
by a modification15
of the method of Jones and
Maurice.16
Baseline AHF was measured in a similar way
without drug or placebo 2 to 13 days before and 10 to 26 days after
LAT-A or -B administration.
Blood–Aqueous Barrier Permeability to Systemic Fluorescein
Topical LAT-A (42 µg) was given to one eye and vehicle to the
opposite eye as described, 1 hour before intravenous injection of
fluorescein Na in the saphenous vein (10 mg/kg in 500–600 µl)
followed by a 4-ml saline flush. The concentration of fluorescein
([fluorescein]) in the cornea and AC was determined by
fluorophotometry 15, 30, 45, 60, 90, 120, 180, and 240 minutes after
fluorescein injection.
Aqueous Humor Protein
Approximately 5.5 hours after LAT-A and vehicle (25% DMSO)
administration or approximately 6.5 hours after LAT-B and vehicle (25%
DMSO) administration, when AHF measurements had been completed, a
sample of AH was obtained under a surgical microscope (Carl Zeiss,
Thornwood, NY), using a 30-gauge needle connected by polyethylene
tubing to a tuberculin syringe. The needle was threaded through the
corneal stroma for approximately 6 mm, then directed into the AC so
that the wound was self-sealing. AH entered the tubing by very gentle
suction with the tuberculin syringe; only occasionally was brief mild
pressure on the cornea with a needle holder required to promote the
initial flow of AH into the tubing through the very thin needle.
Approximately 60 to 80 µl of AH was removed, leaving a shallow but
not completely flattened AC. In separate protocols with different
monkeys, AH was similarly obtained 2 hours after 42 µg LAT-A or 1
hour after 4 µg LAT-B to one eye and vehicle to the opposite eye. The
AH samples were stored at -20°C for up to 24 hours. Protein
concentration in each sample was assayed by the Lowry
method17
18
in duplicate and the result averaged.
Duplicate sets of protein standards containing 0, 1, 3, 5, 10, 20, 40,
or 60 µg bovine serum albumin were assayed by the same method and
results averaged and graphed to give a linear equation that was used to
estimate the protein content in the AH samples. Optical density of each
sample was measured at 660 nm using a spectrophotometer (Spectronic 20;
Bausch and Lomb, Rochester, NY).
Ultrasonic Pachymetry
Corneal thickness was measured using an ultrasonic pachymeter
(model 1000; DGH Technology, Solana Beach, CA). All eyes were examined
by biomicroscopy before baseline measurements. Monkeys were placed
supine in head holders. The central cornea was measured once, and the
peripheral cornea was measured four times midway between the center and
the limbus on the vertical and horizontal axes. For each point in each
eye, two baseline thickness measurements were averaged, and 4.0 µg
LAT-B or 25% DMSO was administered to opposite eyes in the LAT-B
protocol, or 25% DMSO or Bárány’s solution administered
to opposite eyes in the DMSO control protocol. The eyelids were held
open manually during and for 5 minutes after drug or vehicle
administration. Corneal thickness was measured every 30 minutes for 6
hours and again at 24 hours.
Corneal Specular Microscopy
The corneal endothelium of both eyes was photographed using a
specular microscope (provided by Charles J. Koester, Columbia
University, New York, NY) before and 1 hour, 3 hours, 3 days, 7 days,
and 14 days after topical administration of 21 or 42 µg LAT-A to one
eye and vehicle to the opposite eye. Corneal endothelial cell densities
were estimated by counting cells in a reference grid placed over the
photograph. Cells in four separate squares were counted and averaged in
each photograph. All cells completely inside the square and any cells
touching two of the lines making up the square were counted. The number
of cells was multiplied by 100, to compensate for the magnification of
the camera and to determine cell density in cells per square
millimeter. Endothelial cell morphology was evaluated subjectively by a
trained corneal specialist.
Data Analysis
Data are presented as mean ± SEM for n eyes or
animals as indicated. Pre- or post-LAT-A– or -LAT-B–treated eyes
versus contralateral control eyes; post-LAT-A–, post-LAT-B–, or
post-vehicle–treated eyes versus ipsilateral baseline; and
baseline-corrected post-LAT-A– or LAT-B–treated eyes versus control
eyes were compared by using a two-tailed paired t-test.
Differences were compared with 0.0, and ratios were compared with 1.0.
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Results |
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Aqueous Humor Flow and Corneal Endothelial Permeability
The two AHF baselines for individual eyes were similar and were
therefore averaged. The vehicle had no effect on flow rate during any
interval. LAT-A increased apparent AHF by 87% ± 13% (n =
8, P < 0.001; Table 1
) in the first 3 hours, relative to vehicle-treated controls and
adjusted for baseline. During the second 3 hours there were no
significant differences between drug- and vehicle-treated eyes.
Overall, ka increased by 94% ± 9%
(n = 8, P < 0.001), with the increase
perhaps slightly greater during the first 3 hours (106% ± 16%;
P < 0.001) than during the second 3 hours (68% ±
10%; P < 0.001; Table 1
). The relative difference
between the first and second 3-hour intervals was 27% ± 12%
(P < 0.1). After LAT-B administration, there was only
a small, insignificant increase in AHF during the first 3 hours and
little effect on AHF overall (Table 2)
. LAT-B increased ka by 39% ± 5%
(n = 6, P < 0.001; Table 2
) during the
overall 6-hour measurement period, with the greatest increase during
the first 3 hours (58% ± 20%, P < 0.05).
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= 26 ± 8 µm [6% ± 2%],
P < 0.02). Mid-peripheral corneal thickness also
increased 2 to 5 hours after LAT-B administration, with the greatest
increase at 3 hours ( = 32 ± 8 µm [6% ± 1%],
P < 0.005). In eyes treated with DMSO vehicle, corneal
thickness increased from baseline during the first 30 minutes ( = 28 ± 9.6 µm [6% ± 2%], P < 0.025), then
gradually decreased to baseline by 24 hours. By 24 hours, the central
and peripheral corneal thickness in both eyes had returned to baseline.
In the DMSO versus Bárány’s protocol (Figs. 5E 5F
5G
5H)
, there was a variable statistically insignificant tendency for 25%
DMSO to thicken the central cornea initially ( = 14 ± 8.4
µm [4% ± 3%], P < 0.2), followed by slight but
variably significant thinning of both the central and peripheral cornea
( 
5–12 µm [~1–3%]) beyond 2 hours, compared with
the contralateral control eyes receiving Bárány’s
solution.
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Discussion |
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In normally hydrated human corneas, IOP measured by applanation tonometry increases with the corneal thickness.19 20 However, applanation measurements of IOP in edematous corneas may be artifactitiously low, due to the increased sponginess of the edematous cornea. In essence, the epithelium and stroma are being applanated, rather than the entire cornea, and the measurement reflects intracorneal rather than just IOP.21 In this study, LAT-B thickened the central cornea of the live monkey eye, and presumably LAT-A would do the same. However, the LAT-A– or LAT-B–induced IOP reduction is probably not related to changes in corneal thickness, because (1) LAT-B only transiently and slightly thickened the central cornea, with the increase being approximately 47 µm at 3.5 hours (maximal change; only approximately 10% thicker compared with normal monkey cornea) or approximately 26 µm at 6 hours (only approximately 6% thicker than normal); (2) the maximal IOP reduction after LAT-B occurred at 6 hours rather than at 3.5 hours.
LAT-A increased our estimate of AHF by 87% during the first 3 hours after its application. However, what we calculated as increased AHF could also reflect changes in the cornea and blood–aqueous barrier that increase apparent fluorescein clearance without a true change in flow rate. Maus and Brubaker22 recently demonstrated an apparent increase in flow rate of 132%, without a simultaneous increase in IOP, after dilating the pupil with tropicamide and phenylephrine. They concluded that an actual increase in flow of that magnitude was unlikely and that fluorescein may have left the AC through the dilated pupil as well as through conventional outflow.
When we determine AHF rate from the clearance of fluorescein, we assume that the AC and cornea behave as a two-compartment system and that most of the fluorescein leaves by outflow, whereas a fixed amount (representing approximately 10% of normal daytime flow) leaves by diffusion. We also assume that the fluorescence we measure accurately represents the mean concentration of fluorescein in the cornea and AC. Drugs, such as LAT-A, that change structural properties of the anterior segment could violate these assumptions by altering the route and rate of fluorescein clearance as well as our ability to measure it accurately. Thus, we cannot determine whether the apparent increase in flow measured shortly after LAT-A administration represents a true increase in AHF or an overestimate of flow rate, because of limitations in our model of the anterior segment and in our ability to measure fluorescein in this changing environment.
If this system behaves as a two-compartment model, [fluorescein]cornea could increase only after fluorescein passed through the AC after IV injection of fluorescein. However, [fluorescein]AC did not increase, while [fluorescein]cornea increased by 10-fold. Perhaps [fluorescein]AC was partially hidden by quenching from the increased [protein]AC after LAT-A. Possible explanations for the high [fluorescein]cornea may be corneal swelling or edema, changes in reflectance of the corneal endothelium, or a high fluorescein level in the tears. Without additional experiments, we do not have a good explanation for this phenomenon. Nevertheless, the absence of a measurable increase in [fluorescein]AC indicates that blood–aqueous barrier breakdown is at most minimal, consistent with the small and transient increases in [protein]AC measured by the Lowry assay and with the slit lamp findings during IOP measurement in which 4 of 11 LAT-A– and 1 of 8 LAT-B–treated eyes exhibited flare in the AC. The AC flare lasted approximately 24 to 48 hours, far longer than the period of increased [protein]AC determined by Lowry assay. However, this difference does not seem contradictory, because (1) not all monkeys exhibited AC flare; (2) AC flare was found during repeated IOP measurement by applanation tonometry, which necessitated corneal contact, whereas the [protein]AC measurement 5.5 hours after LAT-A was taken after only noncontact fluorophotometry. Weak barrier destabilizing effects of LAT-A and repeated corneal contact tonometry could have been additive. In any event, neither the flare nor the protein increase was substantial. LAT-B seems to have even a less consistent effect on the [protein]AC compared with LAT-A.
Corneal endothelial cells are held together by apical and lateral junctional complexes.23 24 The barrier function of the endothelium depends in part on the state of these junctions.14 25 Cytochalasin B, a fungal metabolite that affects the actin microfilament system by a complex mechanism,26 27 disrupts the apical microfilament network of corneal endothelial cells, causing a change in corneal endothelial morphology and increasing corneal thickness.14 28 29 After topical administration of LAT-A, corneal endothelial cell borders were transiently indistinct. This could represent disruption of cell-cell junctions.30 However, cell shape changes and swelling could simply reorient the cell borders so that they were no longer perpendicular to the incident light, thereby diminishing specular reflected light, and rendering the cell periphery and borders indistinct. The central surface of the swollen cells would still be perpendicular to incident light and would appear as a bright central reflex.31 Biomicroscopy revealed that both LAT-A and -B transiently produced such innumerable small, brightly refractile, granulelike spots, or pseudoguttata, on the corneal endothelium. Specular microscopy after topical LAT-B is needed to clarify whether LAT-B is gentler to the cornea than LAT-A.
The morphologic changes in the corneal endothelium could in turn induce functional changes, as indicated by the increased ka, representing increased corneal endothelial permeability. Similar to AHF, LAT-B–induced ka enhancement was much smaller than that induced by LAT-A, although LAT-B still transiently and mildly increased cornea thickness. Nonetheless, all these morphologic and functional changes in the corneal endothelium induced by LAT-A and -B were apparently reversible, indicating that the cells were not lost.
Collectively, that both LAT-A and B increase outflow facility and reduce IOP suggests their potential as antiglaucoma medications. However, their effects on the cornea, ciliary body, and blood–aqueous barrier are potential safety issues. LAT-B induces a stronger initial facility increase,8 earlier IOP reduction and smaller, less consistent changes in AHF, ka, and [protein]AC than does LAT-A, suggesting that LAT-B may be a better choice. The reason for these differences is not clear yet but could be the different sensitivities of ocular tissues to the two drugs.
Different drug administration strategies may also reduce the side effects. It would be of interest to administer lower concentrations of LAT-A over a longer time to see whether outflow resistance and IOP decrease without affecting the cornea or ciliary body. The high concentration–small volume formulations used in our topical drug protocols to avoid systemic and contralateral effects in the small cynomolgus monkey place the cornea at a disadvantage. The clinician would use lower concentrations in larger volumes, spreading the drug more evenly over the entire corneal surface and exposing the central cornea to a much lower dose. Also, the use of other vehicles, delivery systems, and penetration routes that are less toxic to the cornea could be explored.
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Acknowledgements |
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Footnotes |
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Submitted for publication August 31, 1999; revised January 7, 2000; accepted January 31, 2000.
The University of Wisconsin and the Weizmann Institute of Science hold a patent related to this manuscript. Accordingly, BG and PLK may have a proprietary interest.
Commercial relationships policy: P, C5, Cc1, Cc3, Cc4, Cc6, Cc7, Cc8 (BG, PLK); N (JAP, BT, JWM, WCH).
Corresponding author: Paul L. Kaufman, Department of Ophthalmology and Visual Sciences, 600 Highland Avenue, Madison, WI 53792-3220. kaufmanp{at}mhub.ophth.wisc.edu
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P < 0.1, £P < 0.05,
§P < 0.025, fP < 0.02,
#P < 0.01, and 
P < 0.005).
P <
0.001.
0.0 by the
two-tailed paired t-test for differences:
