Effect of Low-Dose Latrunculin B on Anterior Segment Physiologic Featuresin the Monkey Eye FREE

Latrunculins, macrolides isolated from the marine sponge Latrunculia magnifica, are specific and potent actin-disrupting agentsthat sequester monomeric G-actin, leading to the disassembly of actin filaments.^1- 3 Latrunculins A and B(LAT-A and LAT-B) are the 2 most common latrunculins, which cause reversibledose-dependent and time-dependent destruction of actin bundles and associatedproteins in varieties of cultured cells including human trabecular meshwork(TM) cells.^1- 7 Inliving monkeys, both LAT-A and LAT-B increase outflow facility and decreaseintraocular pressure (IOP).^6,8- 9 Latrunculin-Balso increases outflow facility in the organ-cultured anterior segment ofporcine eyes,⁵ suggesting a direct effect onoutflow resistance in the conventional drainage pathway. The latter has beenconfirmed by a recent morphological study of the TM cells in the live monkeyeye.¹⁰ Because LAT-B, compared with LAT-A,is more potent in increasing outflow facility^6,8 andproduces fewer transient increases in aqueous humor formation, corneal endothelialpermeability, and protein concentration in the anterior chamber(AC),⁹ LAT-B may be a better candidate thanLAT-A as a potential antiglaucoma medication. However, a single dose of 20µL of 500-µM (approximately 0.02%) LAT-B administered topically,which decreases IOP in living monkeys,⁹ stillproduces a transient increase in corneal thickness when applied to the centralcornea as 4 drops of 5-µL volume.⁹ Presumably,multiple treatments with the high concentration of LAT-B might induce moreapparent adverse effects in the cornea.

We hypothesized that repetitive lower concentrations and total dosesin higher solution volumes, spread out over the entire corneal or conjunctivalsurface in the larger human eye, might minimize or avoid toxic effects onthe cornea induced by high concentrations of cytoskeletal drugs without attenuatingtheir effects on outflow resistance.^9,11 Totest this hypothesis, we determined the effects of a single or multiple dosesof 0.005% or 0.01% topical LAT-B on outflow facility, IOP, and/or centralcorneal thickness (CCT) in normotensive monkey eyes. To learn more about thedrug-induced changes in the anterior segment physiologic features, the pupildiameter and accommodation following 0.005% and 0.02% topical LAT-B administrationwere also determined.

Twenty-seven adult normal cynomolgus monkeys (Macacafascicularis) of both sexes, weighing 3 to 8 kg, were studied; 8 forthe tonometry and perfusion protocols, 5 for the pachymetry protocol, and14 for the pupil and accommodation protocols (8 in the 0.02% LAT-B group and6 in the 0.005% LAT-B group). All monkeys contributed 1 eye treated with thedrug and 1 contralateral eye treated with vehicle. All experiments were conductedin accordance with University of Wisconsin-Madison and National Institutesof Health, Bethesda, Md, guidelines and with the Association for Researchin Vision and Ophthalmology Statement on the Use of Animals in Ophthalmicand Vision Research. All monkeys were free of AC cells and flare by slitlampbiomicroscopy when studied. Anesthesia for tonometry or pachymetry was inducedwith intramuscular ketamine hydrochloride (10 mg/kg) and maintained with supplementalintramuscular injections as required (usually 5 mg/kg every 30 to 45 minutes).Anesthesia for AC perfusion or refractometry was induced with intramuscularketamine hydrochloride (10 mg/kg) followed by intravenous pentobarbital sodium(15 mg/kg).

Latrunculin-B was obtained from Sigma Chemical Co (St Louis, Mo) andstored as a 2mM stock solution in dimethyl sulfoxide (DMSO) (Sigma ChemicalCo) at −20°C. Latrunculin-B solutions for topical administrationwere freshly prepared in the Bárány solution¹² with25% DMSO. Twenty microliters of 0.005% (1 µg/20 µL), 0.01% (2µg/20 µL), or 0.02% (4 µg/20 µL) LAT-B were composedof 1.26, 2.53, or 5.00 µL of 2mM LAT-B stock solution in DMSO and 15µL of the Bárány solution, with an additional 3.74 or2.47 µL of DMSO added into the 0.005% or 0.01% drug solution, so thateach drug solution had 25% DMSO. Twenty-five percent DMSO served as a vehiclecontrol. In IOP protocols, the drug or vehicle solution was administered tothe central cornea of opposite eyes of either ketamine-anesthetized (day 1and day 5; 4 × 5-µL drops at each treatment) or fully consciousand manually restrained monkeys (day 2 through day 4; 2 × 10-µLdrops at each treatment) twice daily for 4.5 days at 8 AM and4 PM. Eyedrops were administered at 30- to 60-second intervalswith blinking prevented between drops. Following the 0.01% LAT-B IOP experiment,the monkeys were treated with 0.01% drug and vehicle solution at 4 PM on day 5 and then once (days 6 and 7) or twice (day 8) daily (2× 10 µL at each treatment) for 3 additional days while fully consciousand manually restrained. On day 9, these monkeys were treated again with thesame dose of the drug (4 × 5 µL at each treatment) after receivingketamine anesthesia 2 hours before the AC perfusion. For pachymetry, differentmonkeys were treated with 0.01% LAT-B twice daily for 4.5 days after receivingketamine anesthesia. For refractometry and pupil-diameter measurement, monkeyswere treated with 0.005% and 0.02% LAT-B (4 × 5 µL at each treatment)1 time after receiving ketamine and pentobarbital anesthesia. Administeringthe drug and vehicle solution to fully conscious and manually restrained monkeysin the IOP and outflow facility protocol was designed to reduce any potentialcumulative effect of repeated ketamine administration on IOP or outflow facilityduring the multiple treatments.¹³

Intraocular pressure was determined on day 1 (before and after the firstdose) and on day 5 (before and after the ninth dose) with a minified Goldmannapplanation tonometer,¹⁴ using half-and-halfcreamer solution (Borden Inc, Columbus, Ohio) as the tear film indicator,with the monkey lying prone in a head holder. For each eye, 3 IOP readingswere averaged as a baseline or pretreatment IOP before administration of thefirst or ninth dose of 0.005% or 0.01% LAT-B or vehicle, and single IOP readingswere taken after the drug and vehicle administration hourly for 6 hours. The2-dose IOP protocols were conducted within a successive period of 2 weeksto observe the cumulative dose-response relationship during a short period.The same eyes of the same animals were treated with the drug in the 2 IOPprotocols, with the 0.01% LAT-B experiment performed the week immediatelyfollowing the 0.005% LAT-B experiment. Since there was only a 2-day washoutperiod, some ocular hypotensive effect of the 0.005% dose may have been carriedover to the 0.01% dose protocol, but this cumulative dose-response strategy,which is often used in pharmacological studies, does not affect our overallconclusions.

Total outflow facility was determined by 2-level constant pressure perfusionof the AC with the Bárány mock aqueous humor,¹² usinga 1-needle technique and correcting for internal apparatus resistance.¹⁵ Outflow facility was measured for 90 minutes 2 hoursafter the fifteenth dose of 0.01% LAT-B or vehicle on day 9.

Central corneal thickness was determined by ultrasonic pachymetry (DGH-1000ultrasonic pachymeter; DGH Technology, Inc, Solana Beach, Calif) on day 1(before and after the first dose) and day 5 (before and after the ninth dose).For each eye, 3 readings were averaged as a baseline or pretreatment valuebefore administration of the first or ninth dose of 0.01% LAT-B or vehicle,and single readings were taken after the drug and vehicle administration every30 minutes for 4 hours and then hourly for 2 hours.

Accommodation (difference between baseline and postdrug refraction)was determined with a Hartinger coincidence refractometer (Zeiss-Jena, Jena,Germany). Pupil diameter was measured with Vernier calipers under normal roomlight (350 lux). Baseline refraction and/or pupillary diameter were measured,followed by topical application of 2.5% phenylephrine (stimulates the irisdilator muscle without influencing the iris sphincter and ciliary muscle,^16- 17 facilitating measurement of miosisand accommodation¹⁸). Refraction and/or pupillarydiameter were measured again approximately 30 minutes later, after which 20µL (4 × 5 µL for each treatment) of 0.005% or 0.02% LAT-Bwere administered topically to one eye and vehicle to the other. Refractionand pupillary diameter were determined 85 minutes after LAT-B administration.Five minutes later, approximately 3 mL of pilocarpine solution were infusedintramuscularly in the thigh (1.5 mg/kg) across 10 minutes. Refraction wasdetermined every 5 minutes after pilocarpine infusion until stable, and finalpupillary diameter was then measured. The intramuscular infusion of pilocarpineas described earlier allowed us to measure dose-dependent accommodation duringthe drug administration, where, in effect, time becomes the dose. This methodallowed us to look for differences in the absolute amplitude of the accommodativeresponse and for any leftward or, more likely, rightward shift in the "dose-response"curve of the eye treated with LAT-B relative to the contralateral controleye.^19- 20 Furthermore, systemicadministration assures that both eyes receive the same pilocarpine dose atall times, making the comparison between the eyes at each point still morevalid and precise.

Slitlamp biomicroscopy was performed before drug administration, duringIOP measurement (1, 3, and 6 hours after drug administration), and beforepachymetry and AC perfusion. The integrity of the corneal epithelium and endothelium,the presence of flare or cells in the AC, and the clarity of the lens, werenoted. All animals were free of preexisting ocular abnormalities when studied.

Data are given as mean ± SEM for number of eyes or animals. Predrugor postdrug treated vs contralateral control; postdrug or postvehicle vs ipsilateralbaseline; and baseline-corrected postdrug treated vs control comparisons weremade using the 2-tailed paired t test for differencesvs 0.0 or ratios vs 1.0. The baseline IOP used for the data analysis in the0.005% LAT-B or 0.01% LAT-B protocol was the IOP measured immediately beforethe first treatment of the corresponding dose of the drug or vehicle.

A single dose of 0.005% LAT-B lowered IOP from mean ± SEM 19.3± 0.8 to 16.4 ± 0.7 mm Hg within 6 hours. After adjustment forbaseline and contralateral IOP,²¹ the maximalmean ± SEM hypotension of 2.5 ± 0.3 mm Hg (n = 8; P<.001) occurred at hour 6. Multiple doses (9 doses) of 0.005% LAT-Breduced IOP similar to a single dose but the significant IOP reduction occurredearlier (hour 1 vs hour 3) and the maximal ocular hypotension was slightlygreater (mean ± SEM, 3.2 ± 0.5 mm Hg; P<.001).Intraocular pressure at 16 hours after the eighth treatment (IOP at 0 hourson day 5) in the eye treated with LAT-B was significantly lower than thatin the contralateral control eye (mean ± SEM, −1.4 ± 0.3mm Hg; P<.005) (Figure 1A). A single dose of 0.01% LAT-Blowered IOP from mean ± SEM 18.8 ± 0.7 to 15.7 ± 0.8mm Hg within 6 hours. After adjustment for baseline and contralateral IOP,the maximal mean ± SEM hypotension of 2.7 ± 0.6 mm Hg (n = 8; P<.005) occurred at hour 3. Multiple doses (9 doses)of 0.01% LAT-B induced a greater IOP reduction than a single dose, with themaximal mean ± SEM hypotension of 4.4 ± 0.6 mm Hg (P<.001) at hour 4. The IOP measured before the ninth treatment (IOPat 0 hours on day 5) in the eye treated with LAT-B tended to be lower thanthat in the contralateral control eye (mean ± SEM, −1.7 ±0.7 mm Hg; P = .056). Although the monkeys had notreceived any treatment for 3 days after the ninth treatment with 0.005% LAT-B,the baseline IOP (IOP at 0 hours on day 1) in the eye treated with LAT-B inthe 0.01% LAT-B protocol (Figure 1B)did not return to the level before the first treatment with 0.005% LAT-B (Figure 1A).

Latrunculin B significantly increased outflow facility by mean ±SEM 75% ± 13% (n = 7; P<.005) during theoverall 90-minute postdrug perfusion beginning 2 hours after the 15th treatmentof 0.01% LAT-B. The total number of monkeys was 7 rather than 8 because 1monkey died on day 6 of a disease unrelated to the experiment. In analysisof three 30-minute perfusion periods, the drug increased outflow facilityby mean ± SEM 35% ± 14%, 69% ± 14%, and 100% ±14% in the first, second, and third 30-minute durations, respectively (Table 1). Figure 2 shows that the increase in outflow facility was both timedependent and pressure dependent. There was no facility increase initiallywhen the perfusion was started at the spontaneous IOP of the monkey eye 2hours following administration of LAT-B or vehicle (spontaneous IOP of pentobarbital-anesthetizednormal monkeys is typically <10 mm Hg²²),but a progressive increase occurred across time during perfusion at an elevatedIOP (15 or 25 mm Hg) even though the drug concentration in the AC must havebeen decreasing because of the infusion of drug-free fluid and secretion ofdrug-free aqueous humor into the eye.

On day 1, mean ± SEM baseline CCT was 456.3 ± 17.0 µmin the eye treated with LAT-B and 457.7 ± 18.2 µm in the contralateralcontrol eye. Mean ± SEM CCT after the first treatment varied between454.6 ± 17.2 and 462.4 ± 17.0 µm in the eye treated withLAT-B and between 453.4 ± 15.4 and 458.6 ± 18.7 µm inthe contralateral control eye during 6-hour pachymetry. On day 5, the mean± SEM CCT measured before the ninth treatment was 448.4 ± 17.9µm in the eye treated with LAT-B and 455.9 ± 18.6 µm inthe contralateral control eye. The mean ± SEM CCT after the ninth treatmentvaried between 454.4 ± 16.4 and 462.2 ± 18.2 µm in theeye treated with LAT-B and between 452.2 ± 18.8 and 457.2 ±19.6 µm in the contralateral control eye during 6-hour pachymetry. Collectively,the mean ± SEM CCT in the eye treated with LAT-B was only 0.9 to 8.1µm (P = .08-.82) or 3.1 to 7.1 µm (P = .07-.37) thicker than that in the eye treated withvehicle after 1 or 9 doses of 0.01% LAT-B, after adjustment for ipsilateralbaseline (Figure 3).

Baseline pupil diameters of both eyes in all monkeys were similar (Figure 4Aand C). Twenty-five minutes after phenylephrine administration, both pupilsdilated equally (in the 0.02% LAT-B protocol, mean ± SEM, 7.2 ±0.3 mm vs 7.2 ± 0.3 mm; n = 8; P<.20) (Figure 4A) (in the 0.005% LAT-B protocol,mean ± SEM, 7.0 ± 0.3 mm vs 7.0 ± 0.3 mm; n = 6; P<.40) (Figure 4C).Eighty-five minutes after topical administration of 20 µL of 0.02% LAT-B,the pupils in the eyes treated with LAT-B dilated further relative to thecontralateral controls (to mean ± SEM 8.0 ± 0.3 mm vs 7.0 ±0.4 mm; P<.005) (Figure 4A). However, 85 minutes after 20 µL of 0.005% LAT-B,the pupils in the eyes treated with LAT-B were only slightly larger than thosein the eyes treated with vehicle. When pilocarpine was infused intramuscularlyin the thigh, the control pupils constricted but the pupils treated with 0.02%LAT-B did not (mean ± SEM, 5.6 ± 0.3 mm in controls vs 7.0 ±0.4 mm in eyes treated with LAT-B; P<.001) (Figure 4A). The inhibition of miosis wassubstantial when compared with the pre–LAT-B value (mean ± SEM,7.0 ± 0.4 mm vs 7.2 ± 0.3 mm). The miosis was only slightlyinhibited by 0.005% LAT-B (mean ± SEM, 5.0 ± 0.4 mm in eyestreated with LAT-B vs 4.3 ± 0.3 mm in control eyes).

No significant differences between pilocarpine-induced accommodationin eyes treated with LAT-B vs control eyes were observed initially after 20µL of 0.02% LAT-B (Figure 4B).However, the accommodation plateau in the eyes treated with LAT-B occurredearlier than that in the control eyes (30 vs 40 minutes after the intramuscularpilocarpine). A statistically significant difference between eyes was observedduring the period of 30 to 40 minutes after intramuscular pilocarpine, withthe eyes treated with LAT-B accommodating approximately mean ± SEM2.5 ± 0.5 diopters (D) (approximately 25% ± 8%) less than thecontrols (8.9 vs 11.4 D; n = 5; P<.01) (Figure 4B). The accommodation was only slightlyinhibited by 0.005% LAT-B (Figure 4D).The accommodative amplitude appears greater in the eyes treated with vehiclein the 0.005% LAT-B group compared with the eyes treated with vehicle in the0.02% LAT-B group, which may be owing to the different durations of the measurementin the 2 groups. Other factors might also be involved, such as animal age(not available for some monkeys in the 0.02% LAT-B group), different accommodativeamplitudes in different animals, differences in lag time for systemic bioavailabilityor distribution of the drug, and/or body weight of the animals, that mightaffect muscle mass and therefore distribution of the drug. In any case, thedifference was not statistically significant by the 2-tailed unpaired t test (mean ± SEM, 11.4 ± 1.7 D vs 16.5± 2.2 D; P>.60), and the "different" accommodativeamplitudes did not affect the major conclusions from the data obtained fromcomparison between contralateral eyes of the same monkey.

During IOP measurement, most monkeys had mild punctate corneal epithelialdefects at 3 to 6 hours after the drug administration, but the defects ineyes treated with LAT-B were similar to those in control eyes. Additionally,the punctate corneal epithelial defects seen during tonometry after the firsttreatment on day 1 had disappeared in both eyes of almost all monkeys at approximately16 hours after the eighth dose (before tomometry on day 5). No other abnormalitywas observed in any monkey in any protocol during slitlamp examination. Theheavily pigmented monkey conjunctivas precluded the evaluation of conjunctivalhyperemia.

This study has shown that LAT-B administered topically decreases IOPin normotensive monkeys in a dose-dependent manner, with multiple doses producinggreater IOP reduction than a single dose. This is consistent with many currentclinical and experimental antiglaucoma drugs that have greater effects followingmultiple treatments in both normotensive^21,23 andglaucomatous^24- 25 monkeys. Someocular hypotensive effect of multiple administrations of LAT-B appears tolast more than 16 hours, evidenced by the lower IOP in the eyes treated withLAT-B than in the eyes treated with vehicle at 16 hours after the eighth treatmentin both the 0.005% and 0.01% LAT-B protocols (Figure 1A and B) and by the tendency toward slightly lower baselineIOP in the eyes treated with a drug than in the control eyes 3 days afterthe ninth treatment of 0.005% LAT-B (Figure1B). The latter indicates that the cumulative effect of 0.005% LAT-Bmay affect the apparent IOP response to 0.01% LAT-B given subsequently. However,the IOP measured on day 5 in the 0.01% LAT-B protocol tended to increase 4hours after the ninth treatment, which did not occur in the 0.005% LAT-B protocol.This seems to imply that it is more difficult for the drug to maintain a largerIOP reduction than a smaller one, although a higher dose is used. A more rapidrate of decrease in AC drug concentration due to greater resistance washoutand greater reduction of the pressure gradient between the AC and the Schlemmcanal following the higher dose than the lower dose may account for this phenomenon.Additionally, the same monkeys may have different IOPs or different responsesto the drug on different occasions for a variety of reasons, including anestheticconsiderations. Nevertheless, the IOP at 6 hours after the higher dose wasstill lower than that at 6 hours after the lower dose, which is consistentwith the statement made earlier. In a previous study,⁹ asingle dose of 20 µL of 500-µM (approximately 0.02%) LAT-B maximallydecreased IOP by 3.1 mm Hg, which is slightly greater than the maximal IOPreduction (−2.7 mm Hg) induced by a single dose of 0.01% LAT-B and apparentlysmaller than the IOP reduction (−4.4 mm Hg) induced by multiple dosesof 0.01% LAT-B, in the current experiments. This further indicates that LAT-Bdose dependently decreases IOP and that multiple doses of LAT-B are more effectivethan a single dose. In the present study, 15 treatments with 0.01% LAT-B timedependently and pressure dependently increased outflow facility in the monkeyeye, which, in conjunction with our previous findings,^5,8 suggeststhat LAT-B decreases IOP by reducing outflow resistance in the TM.

In the previous study,⁹ a single doseof 0.02% topical LAT-B also transiently increased the CCT of the monkey eyeby up to 47 µm within 3 hours. Unlike the higher dose studied previously,a single and multiple doses of 0.01% LAT-B administered topically in the presentstudy do not change the CCT. This indicates that the 0.01% concentration ofthe drug does not significantly affect the corneal endothelium. By slitlampbiomicroscopy, 0.01% LAT-B is also less toxic to the corneal epithelium thanthe higher dose studied before.⁹ The LAT-Bdoses used in this study did not produce any additional punctate corneal epithelialdefects in the eyes treated with LAT-B compared with the eyes treated withvehicle. The mild punctate corneal epithelial defects in both eyes, occurring3 to 6 hours after the drug administration, are a common phenomenon duringtonometry in ketamine-anesthetized animals, presumably owing to reduced blinkingunder ketamine anesthesia and frequent IOP measurements. All these seem tosupport our hypothesis from previous studies^9,11 thatrepetitive lower concentrations and total doses in higher solution volumes,spread out over the entire corneal or conjunctival surface, may minimize oravoid toxic effects on the cornea.

A recent morphological study¹⁰ revealedthat LAT-B induces formation of numerous cytoplasmic projections of the subcanalicularcells and massive "ballooning" of the juxtacanalicular region, leading toa substantial expansion of the space between the subcanalicular cell layerand the trabecular collagen beams. Additionally, LAT-B also significantlyincreases the junction-to-junction distance of the inner wall cells of theSchlemm canal,¹⁰ although the increase is notas great as that after the serine-threonine kinase inhibitor H-7.^26- 27 All these structural changes in theTM may be consequent to the drug-induced cellular relaxation and account forthe drug-induced decrease of outflow resistance in the TM. The current physiologicdata indicate that LAT-B dose-dependently relaxes intraocular smooth muscles.This further supports that cellular relaxation could be an important mechanismby which LAT-B decreases outflow resistance in the TM, since H-7, which decreasesoutflow resistance primarily by relaxing the TM,^26- 27 alsorelaxes the iris sphincter in vivo and ciliary muscle strips in vitro.¹⁹ More interestingly, although 0.02% LAT-B appearsto substantially, if not completely, inhibit the miotic response of the monkeyeye to pilocarpine, it only inhibits the accommodative response to the muscarinicagonist by up to 25%. Phenylephrine-induced bilateral mydriasis may allowLAT-B's inhibition of the pilocarpine-induced miosis to be observed more easilybut does not affect the conclusion since phenylephrine was administered bilaterally.The reason for the separation is not clear, but a pharmacokinetic explanationseems plausible.¹⁹ Pilocarpine is a classicalantiglaucoma medication that indirectly increases outflow facility by contractingthe ciliary muscle. However, the induced miosis, which reduces vision especiallyin elderly patients with incipient cataract,²⁸ restrictsits use. Although higher doses of pilocarpine may be more resistant to inhibitionby LAT-B, the relative dissociation of miotic and accommodative responsesto pilocarpine (as used in this study) after LAT-B administration providesa possibility that the combination of a low but still facility-effective topicaldose of pilocarpine with a facility-effective and cornea-safe topical doseof LAT-B may induce a facility increase greater than that induced by eitherdrug alone, without damaging the cornea or constricting the pupil. Furtherstudies are needed to prove this hypothesis.

Collectively, the fact that 0.005% and 0.01% topical LAT-B increaseoutflow facility and/or decrease IOP without adversely affecting the corneasuggests that a low dose of topical LAT-B may have potential as a safe andTM-selective antiglaucoma medication.

Correspondence: Paul L. Kaufman, MD, Department of Ophthalmologyand Visual Sciences, University of Wisconsin-Madison, F4/328 CSC-3220, 600Highland Ave, Madison, WI 53792-3284 (kaufmanp@mhub.ophth.wisc.edu).

Submitted for publication April 9, 2004; final revisionreceived June 2, 2004; accepted June 21, 2004.

This study was supported by grant EY02698 from the National Eye Institute,Bethesda, Md, and grants from the Glaucoma Research Foundation, San Francisco,Calif; Research to Prevent Blindness, New York, NY; the Wisconsin Alumni ResearchFoundation, Madison; and the Ocular Physiology Research and Education Foundation,Madison.

This study was presented at the 140th Annual Meeting of the AmericanOphthalmological Society; May 26, 2004; Hot Springs, Va.

Drs Okka and Tian contributed to this study equally.