The Effect of the Rho-Associated Protein
Kinase Inhibitor, HA-1077, in the Rabbit
Ocular Hypertension Model Induced
by Water Loading
Takaki Fukunaga and Kengo
Ikesugi
Department of Ophthalmology, Graduate School of Medicine, Mie
University, Mie, Japan
Masahiro Nishio
D. Western Therapeutics Institute,
Inc., Nagoya, Japan
Masahiko Sugimoto and
Mikio Sasoh
Department of Ophthalmology, Graduate School of Medicine, Mie
University, Mie, Japan
Hiroyoshi Hidaka
D. Western Therapeutics Institute, Inc., Japan
Yukitaka Uji
Department of Ophthalmology, Graduate School of Medicine, Mie
University, Mie, Japan
ABSTRACT
Purpose: The aim of this study was to investigate the intraocular pressure (IOP)-lowering effect of a new anti-glaucoma drug, the Rho-associated protein kinase (ROCK) inhibitor, HA-1077, in a rabbit ocular hypertension model. Methods: Experiments were carried out in 18 male New Zealand white rabbits, with ocular hypertension induced by water loading. Animals were divided into three groups followed by topical administration of 1 mM, 2 mM, and 3 mM HA-1077 in the left eye. As a control, phosphate buffered saline was administered in the opposite eye. Results: After administration of HA-1077 eye drops, there was a significant time- and dose-dependent decrease of the IOP. While minor conjunctival injection was seen in a few cases, no abnormalities of the anterior chamber or fundus were observed. Conclusions: This is the first report of the effect of the ROCK inhibitor, HA- 1077, on the IOP in an ocular hypertension model. Study results indicated that HA-1077 has a strong IOP-lowering effect.
Keywords: intraocular pressure (IOP); Rho-associated protein kinase (ROCK); ROCK inhibitor HA-1077; tra- becular meshwork; water loading
INTRODUCTION
Glaucoma is a leading cause of permanent blind- ness and is characterized by progressive retinal gan-
Received 11 July 2008; accepted 06 October 2008.
Address correspondence to Dr. Kengo Ikesugi, Department of Oph- thalmology, Graduate School of Medicine, Mie University, 2-174 Edobashi, Tsu, Mie 514-8507, Japan. E-mail: [email protected] u.ac.jp
glion cell death that produces characteristic optic nerve head damage and visual field loss. In normal ten- sion glaucoma, optic nerve damage occurs in pa- tients without any elevation in the intraocular pressure
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(IOP). However, the most common form of glau- coma worldwide is primary open angle glaucoma (POAG), which is often associated with an elevated IOP. Regardless of whether the IOP is within the normal range or not, the major target of glaucoma treatments is a decreased IOP, as the elevation of the IOP is thought to be the major risk factor that causes retinal ganglion cell death in glaucomatous eyes.
At the present time, there are many anti-glaucoma drugs that are used clinically. Prostaglandin F2α (PGF2α ) and its derivatives (e.g., latanoprost, travoprost) are used as anti-glaucoma drugs and have been reported to have a strong IOP-lowering effect.1 After applica- tion of these PGs in the eye, induced increases in the aqueous humor outflow via the uveoscleral pathway are responsible for the alterations in the IOP.
Recently, a novel strategy for the treatment of glaucoma that administers a new drug, Rho-associated protein ki- nase (ROCK) inhibitor, is noteworthy in that it targets the conventional outflow in the eye.2,3 It is theorized that the effect of the ROCK inhibitor occurs via an alter- ation in the contractility and the shapes of the trabecu- lar meshwork cells. This new IOP-lowering mechanism makes a very attractive target for new therapeutic regi- mens, and it is expected that additional effects for other existing anti-glaucoma drugs will be elucidated in the future.
Although several papers have previously reported data on the IOP-lowering effect of ROCK inhibitors in nor-
2–4
mal animal eyes, to the best of our knowledge, there are no reports that have examined the IOP-lowering effect of the ROCK inhibitor HA-1077 in the ocular hy- pertension model.
In this study, in order to investigate this possible new anti-glaucoma drug class, we examined the IOP- lowering effect of the ROCK inhibitor, HA-1077, in the rabbit ocular hypertension model, in which ocular hy- pertension is induced by water loading.
MATERIALS AND METHODS
All animals were treated in accordance with the State- ment for the Use of Animals in Ophthalmic and Vi- sion Research that has been approved by the Associ- ation of Vision Research in Ophthalmology (ARVO). Experiments were carried out in 18 male New Zealand white rabbits, weighing 3.0–3.5 kg. All animals had been previously trained to be handled in a laboratory environment and were maintained under a 12/12-hr light/dark cycle and fed ad libitum. Animals were de- prived of water and food for 24 hr prior to each ex- periment. IOP was measured with a Model 30 Classic
Pneumatonometer (Reichert Inc., Depew, NY, USA). Before each measurement, calibration was checked ac- cording to the manufacturer’s instructions. To induce water loading, rabbits were administered 60 ml of tap water (at 37◦ C) per kilogram of body weight by using a 12-Fr catheter. HA-1077 was dissolved in phosphate buffered saline (PBS) to achieve final concentrations of 1 mM, 2 mM, and 3 mM. Before drug instillation, animals were put under anesthesia using an intramus- cular injection of a mixture of ketamine and xylazine. The depth of anesthesia was enough to ensure the rab- bit tolerated the orogastric intubation and tap water administration. After the baseline IOP (pretreatment pressure) was recorded, 40 µl of each of the HA-1077 concentrations (1 mM, 2 mM, and 3 mM) was topi- cally administered in the left eye of each of the six rabbits in the three different groups. Using the same technique, 40 µl of PBS was administered to the oppo- site eye for use as a control. The time course of this experiment was as follows. First, intraocular pressure was measured prior to the administration of HA-1077. Second, 30 min after the HA-1077 administration, IOP was monitored, followed by the water loading of the rabbits. IOPs were continuously monitored at 30, 60, 90, 120, 180, 240, 300, and 360 min after the water loading. Results are expressed as mean ± standard deviation (SD). Data were analyzed using a paired t-test (JMP Statistical Software, SAS Institute Japan, Tokyo, Japan). Values of p < 0.05 were considered to be statistically significant.
RESULTS
Figure 1 shows the IOP changes that occurred after the HA-1077 administration as compared with the control eyes. In the control group, a statistically significant rise in IOP was found between 30 and 120 min after water loading, with the maximum IOP observed at 30 min after water loading. Compared with vehicle-treated control eyes, there was a significant dose-dependent inhibition of the IOP increase in the HA-1077 treated eyes.
Figure 2 shows the tiIOP. tiIOP is defined as the dif- ference between the IOP of the HA-1077 treated eyes and the IOP of control eyes for each of the timepoints. Figure 3 shows the percentage of the IOP reduction, which was calculated by tiIOP/IOP of the control eyes. For the 1-mM HA-1077 concentration, the maximal IOP reduction (–17.4%) occurred at 120 min, and this IOP reduction was significantly different from the con- trol. For the 2-mM HA-1077 concentration, the maxi- mal IOP reduction (–27.3%) also occurred at 120 min, with a statistically significant reduction observed be- tween 30 and 120 min. After the topical administra- tion of 3-mM HA-1077, the maximal IOP reduction
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Figure 1. Effect of each of the HA-1077 concentrations on the IOP. In each of the rabbit eyes, we topically administered 40 µl of the various HA-1077 concentrations, with the contralateral eyes administered the same volume of a PBS solution for use as the control.
(-46.4%) once again occurred at 120 min, with a statis- tically significant reduction observed between 30 and 360 min. No abnormalities of the anterior chamber or fundus were observed after the topical administration
of HA-1077. Conjunctival injection in eyes treated with 1 mM and 2 mM of HA-1077 was not noted, and only a few cases of 3-mM-treated eyes were slightly injected.
Figure 2. tiIOP was calculated at each timepoint as the difference between the IOP of the HA-1077 treated eyes and the IOP of the control eyes.
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Figure 3. Percentage of IOP reduction was calculated as tiIOP/IOP of the control eye. For every HA-1077 concentration, the maximal IOP reduction percentage occurred at 120 min, with –17.4% observed at 1 mM, –27.3% at 2 mM, and –46.4% at 3 mM. All values were statistically significant: * p < 0.05; ** p < 0.01.
The results demonstrate that there is a strong IOP- lowering effect associated with HA-1077 in the rabbit ocular hypertension model in which hypertension is induced by water loading. These effects are both time- and dose-dependent.
DISCUSSION
Recently, glaucoma, which is characterized by progres- sive retinal ganglion cell death, has become a leading cause of blindness. There are many treatments that are currently being applied, such as the administra- tion of neuroprotective drugs or drugs that lead to an amelioration of the ocular circulation. However, the major objective of glaucoma treatment needs to be the targeting of the mechanism involved in the decrease of the IOP.5 At the present time, there are several drugs known to decrease the IOP via different mechanisms.
In this study we examined a novel anti-glaucoma drug, a ROCK inhibitor, which targets the conventional out- flow in the eye. The effect of the ROCK inhibitor is thought to be associated with alterations in the con- tractility and the shapes of the trabecular meshwork cells.2 Initially, we used the rabbit model that induces ocular hypertension by water loading to evaluate the IOP-lowering effect of the ROCK inhibitor HA-1077.
Since the intraocular pressure does not change after the administration of isosmotic saline, it is theorized that the ocular hypertension induced by the water loading is mainly produced by a passive mechanism that results
6–8
from blood hypoosmolality. A reduction in blood osmolality generates an osmotic gradient between the blood and the aqueous humor, and, thus, there is a passive flow of water into the eye. In addition, a de- crease in the outflow facility of the aqueous humor, which is likely due to a mechanical obstruction of the outflow by hydration of the trabecular meshwork cells ,or due to an increase in the episcleral venous pressure that occurs in response to hydremia, may play a role in the increased intraocular pressure seen after water loading. The aim of the current study was to assess the effect of the ROCK inhibitor on ocular hypertension. We observed a stable increase of the IOP when using this model, similar to that which has been previously reported. Thus, this ocular hypertension model can be considered to be an uncomplicated and useful method for screening anti-glaucoma drugs.
Subsequently, we used this water-loaded rabbit ocular hypertension model to examine the effect of the ROCK inhibitor, HA-1077. In the published report, IOP reduc- tion was observed at 30 min after the administration of HA-1077,9 but in our condition, IOP change was not observed at 30 min after HA-1077 administration. A significant IOP decrease was noted at 30 min after wa- ter loading (at 60 min after HA-1077 administration).
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ROCKs are serine/threonine kinases that have im- portant roles in the fundamental processes of cell migration, cell proliferation, cytoskeletal rearrange- ments, and cell morphology.10 Activation of ROCK by GTP-bound Rho leads to phosphorylation of various target proteins. One of the main substrates of ROCK is the myosin light chain (MLC).11 Phosphorylation of MLC subsequently results in stimulation of the myosin- actin interactions. Increased and decreased MLC phos- phorylation induces contraction and relaxation re- sponses of the cell and influences the formation of actin stress fibers and smooth muscle contraction. Since their discovery, ROCKs have been shown to contribute to several physiological processes by regulating the cy- toskeleton via a variety of different substrates.12,13 HA- 1077, 1-(5-isoquinolinesulfonyl)-homopiperazine, is an antivasospastic compound that has been reported to be a specific inhibitor of ROCK.12-14 As the compound inhibits smooth muscle contraction, it is currently used for the treatment of cerebral vasospasm. In a previously published report, HA-1077 has been shown to be ther- apeutically useful in the treatment of hypertension,15 and ROCK is involved in the pathogenesis of human pulmonary arterial hypertension, HA-1077 might be a novel therapeutic agent that can be used to treat this disease.16 In the eye, it was previously reported that ROCK localizes in the corneal epithelium, and, more re- cently, it has been demonstrated that ROCK is localized in the ciliary muscle tissue and the trabecular mesh- work cells.9 The mechanism responsible for decreas- ing the IOP is theorized to be the same as that which is observed in other cell types, with cell morphology changes causing alterations in the conventional outflow facility.4 Previous reports have indicated that inhibitors of ROCK are capable of causing an increase in the out- flow facility in normal animal eyes.4,9,7 It is believed that these effects are directly related to alterations in the contractility and the shapes of the trabecular mesh- work cells, which, if true, would be a completely new mechanism for decreasing the IOP, thus making the ROCK inhibitor a very attractive alternative as a new anti-glaucoma drug.
While several ROCK inhibitors have been previously reported, we decided to use HA-1077 in this study. HA- 1077 has been shown to inhibit the activity of ROCK along with the activity of other protein kinases.13 In the field of ophthalmic research, several published reports have provided details on the specific effects in animal eyes of another ROCK inhibitor, Y-27632.3,18 HA-1077 exhibits a different protein kinase effect as compared to Y-27632,14 and these ROCK inhibitors have different strengths when acting as an inhibitor of various protein kinases. Since inhibition of each of the protein kinases leads to a reduction of the IOP, this means that not only Rho-kinase but also all other protein kinases might
potentially be able to be used as new anti-glaucoma drugs.
The ROCK inhibitor is a new compound that af- fects the fundamental homeostasis, especially the cy- toskeleton. Since 1995, HA-1077 has been available in Japan as Fasudil and has been used in the treat- ment of cerebral vasospasm after hemorrhage in the subarachnoid space, which is characterized by an in- creased constriction of the cerebral arteries that leads to delayed cerebral ischemia.19-21 In addition, HA- 1077 has also been shown to have beneficial effects in a number of other cardiovascular diseases, including angina pectoris, hypertension, coronary vasospasm, restenosis after percutaneous coronary intervention, and arteriosclerosis.16 Extensive data are available on its safety profile, and all available evidence indicates that this drug is well tolerated in humans. After the top- ical administration of HA-1077 in the current study, we observed a minor conjunctival injection in a few cases, although no other abnormalities of the anterior cham- ber or fundus were seen. Therefore, since we believe that HA-1077 will be well tolerated in human eyes, fur- ther research on the safety of HA-1077, when adminis- tered as a new therapeutic regimen for glaucoma, needs to be undertaken.
Although several papers have previously reported data on the IOP-lowering effect of ROCK inhibitors in nor- mal eyes,2,15,17 to the best of our knowledge, this is the first report of the IOP-lowering effect of the ROCK in- hibitor HA-1077 that has been performed in the ocular hypertension model. As our study demonstrated that HA-1077 has a strong IOP-lowering effect, we believe that new therapies for the treatment of glaucoma based on ROCK inhibitors will be developed in the near fu- ture.
Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.
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