Effect of 5% topical H-7 on intraocularpressure (IOP). A, One dose (group 1, n = 7). B, Seven doses (group 2, n =8). Intraocular pressure data are given as mean ± SEM millimeters ofmercury for the number of animals. Intraocular pressure difference betweeneyes corrected for baseline was tested for differences vs 0.0 by the 2-tailedpaired t test: * indicates P<.05;†, P<.03; ‡, P<.02; §, P<.01; ∥, P<.005; and ¶, P<.001.
Effect of a single dose of 5%H-7 on intraocular pressure (IOP) in monkeys with different baseline (BL)IOP. A, Monkeys with lower BL IOP (group 1b, n = 5; Table 3). B, Monkeys with higher BL IOP (group 4, n = 6). Intraocularpressure data are given as mean ± SEM millimeters of mercury for thenumber of animals. Intraocular pressure difference between eyes correctedfor baseline was tested for differences vs 0.0 by the 2-tailed paired t test: * indicates P<.03;†, P<.01; ‡, P<.005; and dashed line, 16 mm Hg.
Effect of a single dose of 2%H-7 on intraocular pressure (IOP) in the laser-induced unilateral glaucomatousmonkey eyes (group 5, n = 8). Intraocular pressure data are given as mean± SEM millimeters of mercury for the number of animals (eyes). Intraocularpressure difference between day 1 and day 2 was tested for differences vs0.0 by the 2-tailed paired t test: Vehicle indicatesIOP in the eye before and after vehicle on day 1; 2% H-7, IOP in the eye beforeand after H-7 on day 2; *, P<.05; †, P<.005; and ‡, P<.001.
A, Effect of 5 doses of 5% H-7on central corneal thickness in normal monkeys (group 1). The fifth dose ofH-7 or vehicle was given in opposite eyes at time 0. B, Change in centralcorneal thickness. Data are given as mean ± SEM micrometers for thenumber of animals (n = 6 rather than 7 because of the exclusion of 1 monkeyin which a central corneal epithelial defect had occurred in the cage betweenthe second and the third treatments, accompanied by corneal cloudiness andedema). BL indicates baseline; dashed line, no difference between the H-7–treatedeye and the vehicle-treated eye; and slashes, BL was not measured 1 hour orimmediately before the drug administration (it was determined 3-5 days beforethe drug administration. See the "Methods" section). No significant differencebetween eyes was observed at any time.
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Tian B, Wang R, Podos SM, Kaufman PL. Effects of Topical H-7 on Outflow Facility, Intraocular Pressure, andCorneal Thickness in Monkeys. Arch Ophthalmol. 2004;122(8):1171–1177. doi:10.1001/archopht.122.8.1171
To determine if low concentrations of H-7 (1-[5-isoquinoline sulfonyl]-2-methylpiperazine) topically applied to the eye increases outflow facility and decreasesintraocular pressure (IOP) without affecting the cornea in monkeys, and toevaluate if the effect of H-7 on IOP is pressure dependent.
Single or multiple doses of 5% H-7 or vehicle (20 µL) were administeredtopically to opposite eyes of normal monkeys. A single dose of 2% H-7 or vehicle(50 µL) was administered to the glaucomatous eye of monkeys with laser-inducedunilateral glaucoma, with vehicle on day 1 and H-7 on day 2.
In normotensive eyes, 1 dose of 5% H-7 maximally decreased IOP by amean ± SEM of 2.5 ± 1.0 mm Hg (−16.7% ± 5.5%) at3 hours. Higher baseline IOP and repeated dosing were associated with greaterIOP reduction. Outflow facility was increased, but central corneal thicknesswas not affected. In glaucomatous eyes, 1 dose of 2% H-7 maximally decreasedIOP by a mean ± SEM of 5.8 ± 0.6 mm Hg (−16.9% ±1.6%) at 2 hours.
Five percent H-7 increases outflow facility and decreases IOP, but doesnot affect corneal thickness. Multiple doses of H-7 induce greater reductionof IOP than a single dose. The effect of H-7 on IOP may be pressure dependent.
Multiple topical treatments with low doses of H-7 or analogues may substantiallyreduce outflow resistance in the hypertensive eye without meaningfully affectingthe cornea.
The serine-threonine kinase inhibitor H-7 (1-[5-isoquinoline sulfonyl]-2-methylpiperazine) inhibits actomyosin-driven contractility, probably by inhibitingmyosin light chain kinase or rho kinase. This leads to deterioration of theactin microfilament system and perturbation of its membrane anchorage andto loss of stress fibers and focal contacts in many types of cultured cells,including human trabecular cells.1-7 H-7administered intracamerally or topically to living cynomolgus monkeys increasesoutflow facility and decreases intraocular pressure (IOP)5 bya mechanism independent of the ciliary muscle, presumably acting directlyon the trabecular meshwork (TM).8 A morphologicalstudy9 of the TM in the live monkey eye indicatesthat H-7 reduces TM cell contractility, thereby "relaxing" the trabecularoutflow pathway, expanding the draining surface, and facilitating flow throughthe meshwork. Because the effect of H-7 on outflow resistance is pressuredependent,5 it is assumed that H-7 may be moreeffective in the glaucomatous eye with elevated IOP. However, 20 µLof 400 mM H-7 (approximately 15%) administered topically, which decreasesIOP in living monkeys,5 also produces transientchanges in corneal endothelial cells and corneal thickness when applied tothe central cornea as 4 drops of 5-µL volume.10 Presumably,multiple treatments with the high concentration of H-7 may induce more apparentadverse effects in the cornea.
We hypothesized that repetitive lower concentrations and total dosesin higher volumes, spread out over the entire corneal or conjunctival surfacein the larger human eye, might minimize or avoid corneal toxicity inducedby high concentrations of H-7 without attenuating its effect on outflow resistance,especially in the hypertensive eye.10 To testthis hypothesis, we determined the effects of single or multiple doses of5% topical H-7 on outflow facility, IOP, and corneal thickness in normotensivemonkey eyes, as well as the effect of a single dose of 2% topical H-7 on IOPin laser-induced ocular hypertensive monkey eyes.
Thirty-two adult cynomolgus monkeys (Macaca fascicularis) of both sexes, weighing 2 to 5 kg, were involved in this study. Allexperiments were conducted in accord with the University of Wisconsin–MadisonMedical School and Mount Sinai School of Medicine institutional animal careand use committees, with the National Institutes of Health guidelines, andwith the Association for Research in Vision and Ophthalmology Statement onthe Use of Animals in Ophthalmic and Vision Research. Five percent H-7 wasused in 24 normotensive monkeys studied at the University of Wisconsin–MadisonMedical School as 4 groups (groups 1-4); 3 monkeys were included in both group1 and group 3, and 2 monkeys were included in both group 1 and group 4. Monkeysin group 3 had undergone prior anterior chamber (AC) perfusions, but not withinthe preceding 5 to 6 weeks. Monkeys in group 4 had higher baseline IOP (meanIOP, 16 mm Hg) under ketamine hydrochloride (hereafter ketamine) anesthesia in both eyes compared with most of the normalcynomolgus monkeys. Two percent H-7 was used in 8 monkeys with unilateralocular hypertension (mean IOP, 30-35 mm Hg) induced by repeated argon or diodelaser photocoagulation of the TM11 studiedat the Mount Sinai School of Medicine as group 5. The glaucomatous monkeyshad not received any treatment for at least 2 weeks before the testing. Allmonkeys were free of aqueous humor cells and flare as assessed by slitlampbiomicroscopy. In normal monkeys, anesthesia for tonometry or pachymetry wasinduced with intramuscular ketamine (10 mg per kilogram of body weight) andmaintained with supplemental intramuscular injections as required (usually5 mg/kg every 30-45 minutes). Anesthesia for AC perfusion was induced withintramuscular ketamine (10 mg/kg), followed by intravenous pentobarbital sodium(15 mg/kg). In glaucomatous monkeys, anesthesia for tonometry was inducedwith intramuscular ketamine (2-5 mg/kg), with 1 drop of 0.5% proparacainehydrochloride applied topically 5 minutes before tonometry.
H-7 was obtained from Sigma-Aldrich Corporation (St Louis, Mo). Fivepercent H-7 was freshly dissolved for topical administration in isotonic sodiumchloride alone or with 25% dimethyl sulfoxide (1 mg/20 µL); isotonicsodium chloride with (group 3) or without (groups 1, 2, and 4) 25% dimethylsulfoxide served as a vehicle control. The pH (tested by pH paper) of 5% H-7in isotonic sodium chloride was approximately 2 to 3 (adjustment of pH withsodium hydroxide reduces solubility of H-7). To increase the pH of 5% H-7,we dissolved the drug in 25% dimethyl sulfoxide with isotonic sodium chloridein group 3. Twenty-five percent dimethyl sulfoxide seemed to improve the solubilityof H-7 when the pH was slightly elevated (eg, approximately 3-4). Two percentH-7 was freshly prepared in isotonic sodium chloride (1 mg/50 µL), andisotonic sodium chloride served as a vehicle control. Administrations of H-7or vehicle in the different groups are shown in Table 1. For normal monkeys, H-7 or vehicle (4 × 5 µL)was administered topically to the central cornea of the supine monkey underketamine anesthesia for tonometry and pachymetry or was administered topicallyto the superior cornea of the prone monkey under pentobarbital sodium forperfusion.5 To reduce any potential cumulativeeffect of repeated ketamine anesthesia on IOP or outflow facility during themultiple treatments, some doses of H-7 or vehicle were administered topicallyto fully conscious and manually restrained monkeys, with fewer and largerdrops (2 × 10 µL) applied because of the limited cooperative periodfrom conscious monkeys. For glaucomatous monkeys, H-7 or vehicle (2 ×25 µL) was administered topically to the central cornea in a mannersimilar to that in ketamine-anesthetized normal monkeys.
In normal monkeys, IOP was determined with a minified Goldmann applanationtonometer,12 using fluorescein as the tearfilm indicator, with the monkey lying prone in a head holder. For each eye,3 IOP readings were averaged as a baseline before administration of H-7 orvehicle, and single IOP readings were taken after the drug or vehicle administrationat different time points (Table 1).In glaucomatous monkeys, IOP was determined with a calibrated pneumatonometer(model 30 classic; Mentor Inc, Norwell, Mass).
Total outflow facility was determined in normotensive monkeys of group3 by 2-level constant pressure (approximately 15 or 25 mm Hg) perfusion ofthe AC with Bárány mock aqueous humor,13 usinga 1-needle technique and correcting for internal apparatus resistance (Table 1 and Table 2).14
Central corneal thickness was determined in normal monkeys (group 1)by ultrasonic pachymetry (DGH-1000 ultrasonic pachymeter; DGH Technology,Inc, Solana Beach, Calif). For each eye, 3 readings taken 3 or 5 days beforethe first dose of 5% H-7 or vehicle were averaged as a baseline. A singlevalue was taken before the fifth treatment; after the fifth treatment, measurementswere taken every 30 minutes for 4 hours and then hourly for 2 hours.
In normal monkeys, slitlamp biomicroscopy was performed before drugadministration, during IOP measurement (1, 3, and 6 hours after drug administration),and before pachymetry and AC perfusion. In glaucomatous monkeys, slitlampexamination was performed before and 1, 3, and 5 hours after drug administration.The integrity of the corneal epithelium and endothelium, the presence of flareor cells in the AC, and the clarity of lens were noted. All normal animalswere free of preexisting ocular abnormalities when studied. All glaucomatousmonkeys were free of aqueous humor flare and cells when studied.
Data are given as mean ± SEM for the number of eyes or animals.In normal monkeys (groups 1-4), comparisons were made using the 2-tailed paired t test for differences vs 0.0 or for ratios vs 1.0 (predrugor postdrug treated vs contralateral control, postdrug or postvehicle vs ipsilateralbaseline, and baseline-corrected postdrug treated vs control). In glaucomatousmonkeys (group 5), IOP difference between day 1 (vehicle) and day 2 (H-7)in the ocular hypertensive eye was tested by the 2-tailed paired t test.
Intraocular pressure changes from 1 to 6 hours after H-7 or vehiclein the different groups are shown in Figure1, Figure 2, and Figure 3. Maximal IOP reductions after H-7in the different groups are shown in Table3.
In group 3 (normal monkeys), H-7 significantly increased outflow facilityby 77% ± 32% (n = 8, P<.05) during theoverall 90-minute perfusion beginning 1½ hours after the seventh dose,adjusted for outflow facility before the seventh dose (baseline) and contralateralcontrol eye washout (Table 1 2).Outflow rate at 25 mm Hg during baseline or postdrug measurement was higherthan that at 15 mm Hg in the H-7–treated eye and the contralateral controleye. The increase of outflow rate in the H-7–treated eye at 25 mm Hgwas similar to that at 15 mm Hg, after adjustment for baseline and contralateralcontrol eye washout.
Central corneal thicknesses at baseline and before the fifth treatmentwere 456.3 ± 15.6 and 469.8 ± 15.7 µm, respectively, inthe H-7–treated eye and 449.7 ± 14.8 and 456.2 ± 15.1µm, respectively, in the contralateral control eye. The central cornealthickness after the fifth treatment varied between 455.8 ± 17.9 and471.0 ± 15.6 µm in the H-7–treated eye and between 449.2± 13.2 and 461.7 ± 14.4 µm in the contralateral controleye during 6-hour pachymetry. No significant difference in the central cornealthickness between eyes was observed at any indicated time (P>.60), after adjustment for ipsilateral baseline (Figure 4).
During IOP measurement in normal monkeys, most monkeys in groups 1,2, and 4 had mild punctate corneal epithelial defects (PCEDs) in both eyes,but the defects in H-7–treated eyes were slightly more apparent thanin control eyes (Table 4). Twomonkeys (one in group 1 and another in group 2) that had shown continuousnystagmus under ketamine anesthesia during IOP measurement had a small epithelialdefect in the central cornea of the H-7–treated eye at the end of tonometry.The monkey in group 2, but not the one in group 1, had corneal cloudinessand edema 2 days later. Another monkey in group 1 that had shown some PCEDsduring tonometry on day 1 had a central corneal epithelial defect before thethird treatment on day 2 (presumably occurring in the cage) and corneal cloudinessand edema on day 3. However, the cornea was normal for all monkeys approximately16 hours after the sixth dose of H-7 in group 2 (before tonometry) and group3 (before AC perfusion). In addition, the PCEDs seen during tonometry earlierhad disappeared or significantly decreased in both eyes of most monkeys approximately16 hours after the fourth dose (before pachymetry) in group 1. No other abnormalitywas observed in any monkey in any protocol during slitlamp examination ingroups 1, 2, and 4. In glaucomatous monkeys (group 5), 1 of 8 ocular hypertensiveeyes had mild PCEDs 3 and 5 hours after drug administration.
This study showed that single and multiple doses of 5% H-7 decreaseIOP in normotensive monkeys, with multiple doses producing greater IOP reduction.This additive effect of multiple treatments occurs with many antiglaucomadrugs in normotensive15,16 andglaucomatous17,18 monkeys. Inthe present study, IOP reduction after H-7 in normal monkeys with higher baselineIOP (mean IOP, 17 mm Hg) was greater than that in monkeys with lower baselineIOP (mean IOP, approximately 13 mm Hg), consistent with higher outflow rateat 25 mm Hg than at 15 mm Hg during perfusion. This, in conjunction with previousfindings,5,9,19 suggeststhat H-7 decreases IOP by reducing outflow resistance in the TM and that theeffect of H-7 on outflow resistance is pressure dependent. The similarityof outflow rates as defined by the double ratios ([H-7/Baseline] / [Vehicle/Baseline])at 25 and 15 mm Hg indicates that the perfusion-induced resistance washoutduring bilateral baseline and contralateral postvehicle measurements is alsopressure dependent. One can reasonably hypothesize that still lower concentrationsof H-7 in the hypertensive eye may show the same or stronger effect as 5%H-7 does in normotensive monkeys. As previously hypothesized,10 lowerconcentrations and total doses in higher volumes might minimize or avoid cornealtoxicity induced by high concentrations of H-7.
To test these hypotheses, a single dose of 50 µL of 2% H-7 wasapplied in laser-induced hypertensive eyes. Fifty microliters of 2% H-7 inglaucomatous eyes produced a similar IOP reduction compared with 20 µLof 5% H-7 in the normotensive eyes of group 1 (−16.9% vs −16.7%).However, compared with the normotensive eyes of group 4, which had higherbaseline IOP, the maximal IOP reduction in the glaucomatous eyes after 50µL of 2% H-7 was smaller than that in the normal eyes after 20 µLof 5% H-7 (−16.9% vs −24.8%). Although 50 µL of 2% H-7 containsthe same amount of H-7 (1 mg) as 20 µL of 5% H-7, the former may beless effective than the latter when administered topically in the monkey eyebecause the 50-µL volume far exceeds the capacity of the monkey's cul-de-sac,so that some of the drug may overflow during the application. This may reducethe amountof drug penetrating into the eye and, in turn, the concentrationof the drug in the AC. This speculation is in agreement with previous studies20-22 in which the bioavailabilityof topical medications can be increased by decreasing the volume of eyedrops.In addition, laser-induced structural changes in outflow pathway may causethe absence of an expected baseline pressure–dependent IOP reductionin the glaucomatous eye. Based on previous studies,23-26 laserphotocoagulation of the TM induces scar tissue formation and decreases normalcells in the outflow pathway, which increases the tissue's stiffness and limitsits relaxation after H-7. Therefore, although most clinical and investigationalocular hypotensive agents are more effective in the laser glaucomatous monkeymodel than in the normotensive monkey eye, it may not be so for cytoskeletaldrugs (eg, H-7) that increase outflow facility by inhibiting cellular contractilityin the TM and inner wall of Schlemm canal. Given the fact that 5% H-7 producedgreater IOP reduction in group 4 than in group 1, H-7 might produce greaterocular hypotension in non–laser-induced glaucoma models in the monkeyeye (eg, steroid-induced glaucoma27-29)or in glaucomatous human eyes in which anatomy is relatively undisturbed.Further studies are needed to clarify this issue.
Unlike a single topical dose of approximately 15% H-7 (2.9 mg/20 µL),10 5 doses of 5% H-7 (1 mg/20 µL) given topicallydid not significantly increase the central corneal thickness. This indicatesthat the 5% concentration of the drug does not significantly affect the cornealendothelium. As assessed by slitlamp biomicroscopy, 5% H-7 is also less toxicto the corneal epithelium.10 Mild PCEDs inboth eyes are a common phenomenon during tonometry in ketamine-anesthetizedanimals and may be due to reduced blinking under ketamine anesthesia and frequentIOP measurements. However, compared with the contralateral control eyes, theslightly more apparent PCEDs in the H-7–treated eyes indicate that 5%H-7 may still mildly affect the corneal epithelium in living monkey eyes.Two monkeys that had a central corneal epithelial defect during tonometryhad had continuous vertical nystagmus after ketamine anesthesia. This suggeststhat 5% H-7 could render the cornea more susceptible to mechanical damage(eg, frequent contact by the moving eye with the tip of the Goldmann applanationtonometer). Similarly, touching the eye by hand because of an uncomfortablefeeling following drug treatment and tonometry may be responsible for onemonkey's developing a central corneal epithelial defect in its cage. However,this study shows that multiple doses of 5% H-7 seem to have no effect on thecornea as seen by slitlamp examination when the cornea is untouched. In addition,a single dose of 2% H-7 (1 mg/50 µL) did not apparently affect the cornealepithelium as assessed by slitlamp examination in glaucomatous monkeys, comparedwith a single dose of 5% H-7 (1 mg/20 µL) in normal monkeys. This seemsto support the hypothesis from a previous study10 thatrepetitive lower concentrations and total doses in higher volumes, spreadout over the entire corneal or conjunctival surface, may minimize or avoidcorneal toxicity.
Correspondence: Paul L. Kaufman, Department of Ophthalmology andVisual Sciences, University of Wisconsin–Madison Medical School, RoomF4/328, Clinical Science Center, Mail Stop 3220, 600 Highland Ave, Madison,WI 53792-3284 (firstname.lastname@example.org).
Submitted for publication May 28, 2003; final revision received November24, 2003; accepted January 21, 2004.
This study was supported by grant EY02698 from the National Eye Institute,Bethesda, Md (Dr Kaufman); core grant EY01867 from Mount Sinai School of Medicine;Glaucoma Research Foundation, San Francisco, Calif (Dr Kaufman); Researchto Prevent Blindness, New York, NY (Drs Kaufman and Podos); University ofWisconsin–Madison Alumni Research Foundation (Dr Kaufman); Ocular PhysiologyResearch and Education Foundation, Madison (Dr Kaufman); and May and SamuelRudin Family Foundation, New York (Dr Podos).