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Topical Isopropyl Unoprostone for Retinitis Pigmentosa: Microperimetric Results of the Phase 2 Clinical Study

Introduction The purpose of this study was to determine whether topical 0.15% isopropyl unoprostone (IU), a BK-channel activator, could improve or maintain the central retinal sensitivity in patients with middle- to late-stage retinitis pigmentosa (RP). IU was approved for glaucoma and ocular hypertension in 1994. The drug re-profiling strategy is one of the effective ways to develop safe drugs for patients with RP. Methods A randomized, double-blind, and placebo-controlled phase II safety/efficacy trial was conducted. One hundred and nine patients with middle- to late-stage RP having a visual acuity of ≥0.5 were studied at six ophthalmological centers in Japan. The treatments of IU/day were divided into three groups: placebo group; two-drop group; and four-drop group for 24 weeks. The primary outcome measure was changes in the retinal sensitivity from baseline in the central 2° determined by MP-1 microperimetry (MP-1, Nidek, Japan). The secondary outcomes were changes in best-correct visual acuity, contrast sensitivity, retinal sensitivity of the central 10° by MP-1, mean deviation (MD) by a Humphrey field analyzer (HFA; Carl Zeiss Meditec, Dublin, CA, USA) 10-2, and the Visual Functioning Questionnaire 25 (VFQ-25) questionnaire scores. Results There was a tendency for a dose-dependent responsiveness in retinal sensitivity in the central 2°, MD, and total VFQ-25 score after 24 weeks of IU instillation by a simple linear regression analysis. A stratified analysis showed a significant dose-dependent responsiveness of the 2° central retinal sensitivity in more advanced patients (P = 0.028). The number of patients having a ≥4 dB decrease in the primary outcome measure was significantly fewer in the four-drop group than in the placebo group (P = 0.02). No adverse reactions were observed. Conclusions A higher dose of IU can delay progression of the central retinal sensitivity decrease through an improvement of retinal sensitivity.


INTRODUCTION
Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal degenerative diseases characterized by night blindness and progressive decrease of peripheral vision [1,2]. At more advanced stages, the central vision is also reduced. The worldwide prevalence of RP is about 1 in 4,000 for a total of more than 1 million affected individuals [1,2], and over 50 genes have been cloned or mapped for RP.
RP typically begins with a degeneration of the rods followed by a progressive and irreversible death of the cones, which accounts for the loss of central vision and complete blindness. It appears that cones are dependent on rods for survival [3] and once the rods die, death of the cones is inevitable. Most patients with RP visit ophthalmologists in the middle to late stages of the diseases where most rods die and cones start deteriorating. Thus, prevention of the secondary degeneration of cones is an important goal for preserving central vision in patients with RP.
The results of clinical trials, including nutritional supplementation trials have shown that photoreceptor degeneration can be slowed [4][5][6][7][8]. In a recent phase I clinical trial, patients with severe RP had encapsulated ciliary neurotrophic factor-secreting cells implanted intraocularly [9], which this led to an improvement in vision. The results of two clinical studies with RPE65 gene replacement showed that there was improvement of visual acuity and retinal sensitivity in patients with Leber's congenital amaurosis [10,11]. However, long-term efficacy, immune responses, and complications associated with the use of vectors to deliver the genes need to be considered when gene therapy is used. Although cell or gene replacement therapy may be the ultimate method to rescue photoreceptor cells in patients with RP, it is still reasonable to test small molecule drugs with known neuroprotective properties and good safety profiles. This drug re-profiling strategy could be effective in developing safer drugs for patients with RP. It must be remembered that the drugs will need to be used throughout the patient's lifetime. From this point, four exploratory clinical studies in patients with RP have been conducted: intravitreal implantation of brimonidine (NCT00661479), oral valproic acid [7] (NCT01233609 and NCT01399515), oral Ca 2? -channel blocker [8], and topical 0.12% isopropyl unoprostone (IU; Rescula Ò , R-tech Ueno, Tokyo, Japan) [12]. These mutationindependent therapies could possibly slow or stop photoreceptor degeneration without directly treating the original abnormality. IU is a metabolized form of prostaglandin F 2a , a chemically synthesized docosanoid (22-carbon basic skeleton), and 0.12% IU was approved for clinical use in Japan in 1994, and 0.15% IU was approved in the United States in 2001. IU is used to treat eyes with glaucoma or ocular hypertension [13], and clinical studies showed that topical IU led to an increase in retinal and choroidal blood flow in patients with glaucoma [14,15]. The mechanism of action of IU is as a BK-channel or maxi-K channel activator [16][17][18] and not as a prostaglandin F(FP) receptor agonist [17,18]. BK channels are potassium channels that reach an activation threshold only during depolarization and/or at high intracellular Ca 2?
concentrations [17,19]. Laboratory experiments showed that an intravitreal injection of IU in rats protected the photoreceptors against lightinduced damage in a dose-dependent manner [20], and retinal ganglion cells against endothelin 1 (ET-1)-induced neuronal injury through ERK phosphorylation [21]. An in vitro study demonstrated that IU had anti-apoptotic activity on retinal neuroglial progenitor R28 cells of rats [22].  [12].
To treat patients with RP throughout their lives, IU should be considered because it has a good safety profile [25] and has neuroprotective properties [17][18][19][20][21][22] (e.g., anti-oxidative, antiapoptosis, anti-light induced injury, and anti-ET-1-induced neuronal injury properties). In addition, although the primary lesion in RP results in apoptosis of the photoreceptor cells due to a gene mutation, secondary factors are considered to damage photoreceptor cells, in particular cone cells [3]. One of the factors might be reduced choroidal blood flow, which has been reported to be present in patients with RP [26][27][28]. In a rat model, impairment of choroidal circulation was probably involved in delayed cone death after light toxicity [29]. The authors suggested that improving choroidal circulation might preserve the cones and remaining rods in patients. An increase in the plasma level of ET-1, which is related to a decrease in foveal choroidal blood flow, has been reported in some patients with RP [30,31]. Multiple instillations of IU were shown to partially block the ET-1-induced vasoconstriction of the human choroidal vessels [32].
In patients with the later stage of RP, preservation of the secondary death of central cones is an important goal to maintain daily lives. The purpose of this study was to determine the safety and efficacy of 0.15% topical IU for the treatment of patients with the middle to late stage of typical RP whose central cones started to deteriorate. The clinical trial was conducted with a study design of a randomized, double-blind, placebo-controlled, parallel assignment, six-center, phase II study of 6 months' duration.

Study Objectives
The primary endpoint was changes of the mean retinal sensitivity of the central four spots    Decimal visual acuity of 0.5 or more 3. Results of a Goldmann visual field test show a visual field abnormality of a moderate to severe stage 4. The difference in mean retinal intensity of the four retinal points is up to 2 dB between two measurements conducted within a month using an MP-1, and any of the value ranges from 6 to 16 dB. If the criteria are not met with two measurements, a third measurement is made within a month after the second measurement, and either of the two values with a difference of up to 2 dB is within the range 6-16 dB 5. The difference in the MD is up to 3 dB in two reliable measurements a of retinal sensitivity made within a month using a Humphrey visual field analyzer (10-2, SITA-Standard), and visual field abnormality is recognized in these two measurements. If the criterion is not met with the two measurements, a third measurement is made within a month after the second measurement. If the third measurement is reliable, the difference of the value from the first or second measurement is up to 3 dB, and visual field abnormality is recognized in the two measurements Ocular exclusion criteria (applies to the eye evaluated) 1. Under treatment for glaucoma or ocular hypertension 2. Either eye enucleated or eviscerated 3. Cone-rod dystrophy with primarily impaired cone function 4. History of other ocular disease that can confound the outcome of the study (e.g., diabetic retinopathy, uveitis) 5. Complicated by remarkable cystoid macular edema, epimacular membrane, or macular traction syndrome, diagnosed by OCT 6. Complicated by a nuclear cataract of moderate grade (grade 3, Emery classification), an anterior subcapsular cataract, or a posterior subcapsular cataract, that may seriously affect vision 7. 0.12% isopropyl unoprostone used within the past 1 year 8. Any of the following drugs used within 1 month before informed consent is obtained. Steroids, drugs that dilate the blood vessels, and dark adaptation improving drugs (Xantofyl palmitato b )

Examination Procedures
The CSV-1000E (Vector Vision Co, Greenville, OH, USA) was used to determine BCVA by the Early Treatment Diabetic Retinopathy Study method and the spatial contrast sensitivity. Spatial frequencies of 3, 6, 12, and 18 cycles/ degree were studied; each spatial frequency included eight different levels of contrast.
From the data obtained by CSV-1000E, the area under the log contrast sensitivity function (AULCSF) was calculated by the method of Applegate and associates [34]. Microperimetric

RESULTS
A signed informed consent form to participate in the clinical study was obtained from 180 patients with RP. Sixty-eight patients did not to meet the inclusion criteria during the screening stage, and 112 patients were studied.
These 112 patients were randomized into three groups: 35 in the placebo group; 39 in the twodrop group; and 38 in the four-drop group. The eye drops were discontinued in four patients during the course of the study; three from the placebo group, and one from the two-drop group, and 108 patients completed the study.
The reasons for discontinuation of the eye drops were patients' request in one of the placebo group, and the onset of adverse events in three patients; two in the placebo group, and one in the two-drop group.
The efficacy of IU was determined from 109 patients; 108 who completed the 24-week protocol and one from the placebo group who was examined at week 16 but was not able to complete the study due to adverse events.  These observations suggested that improvements in the placebo group were natural fluctuations and associated with better retina sensitivity before the beginning of IU. A stratified analysis was made on 109 patients by dividing them into two groups with a cutoff value of 12.5 dB, the median sensitivity, before beginning the IU (Fig. 4). A dose-dependent responsiveness was found in the three groups with the mean retina sensitivity of less than 12.5 dB (P = 0.028, simple linear regression analysis) (Fig. 4b). An intergroup comparison showed that the value of the improvement in the four-drop group was significantly greater than that in the placebo group (P = 0.019, Williams' test). The four-drop group had a mean improvement of 2.1 (1.2 ? 0.9) dB over the placebo group. With a mean retina sensitivity of 12.5 dB or more all groups did not have a significant increase in their retinal sensitivities (Fig. 4a).
The changes in secondary endpoints in the three groups as a function of the dose of IU are    Table 7.
A dose-dependent reaction was not evident in any of the items except eye irritation. However, there is no significant difference between the two-drop and the four-drop groups. The most frequently observed adverse event was eye irritation. The highest incidence was observed in the two-drop group (53.8%), while it was 11.4% in the placebo group and 44.7% in the four-drop group. The next most frequent adverse event was punctate keratitis, which was observed most frequently in the placebo group (17%), and the incidence was not significantly different between the two-drop group and the four-drop group (10%).    that the reproducibility of data obtained by either an MP-1 or HFA by making two to three measurements within a month for the baseline data.

DISCUSSION
In this clinical trial, the authors validated changes in the mean retinal sensitivity of the 2°b y a MP-1 as the primary endpoint from three aspects: the results of the previous pilot study [12]; the central 2°area reportedly having the best reproducibility and statistical significance in a MP-1 [48]; and a higher correlation between visual acuity and retinal sensitivity in the central 2°in HFA 10-2 [42]. The authors selected a range of mean retinal sensitivity in the central 2°from 6 to 16 dB as an inclusion criterion. The central retinal sensitivity was reported to be 19.7 ± 0.8 dB ranging 16-20 dB in normal subjects [49], and 18.6 ± 1.5 dB in normal elderly individuals [48]. Accordingly, the authors set the upper limit to 16    These findings suggested that IU was more effective in patients with RP whose central retinal sensitivities were more decreased, namely those at late-or advanced-stage RP.
The four-drop group showed an average improvement of 1.24 (0.13 ? 1.11) dB over the placebo group. This is a relatively small change because all the values were averaged. When looking at the value of each patient with RP in Fig. 3, however, the number of patients who had a worsening of -4 dB or more in retinal sensitivity of the central 2°was significantly fewer in the four-drop group than in the placebo group (P = 0.02). Clinically, this degree of change should indicate that there was a slowing in the progress of the disease process in the 24-week observation period.
What might be the mechanism for the improvement of mean retinal sensitivity in the central 2°after 24 weeks of topical IU? The route of IU to the posterior ocular tissues is by a direct diffusion through the eye [50] or through the ocular blood circulation through the nasal and conjunctival vascular systems. The human choroidal blood flow improved after ocular instillation of three and four drops of 0.12% IU/time after the blood flow was decreased by the injection of ET-1 intravenously [32]. In this study, the ocular instillation of four drops of 0.15% IU/day in both eyes, in total eight drops/ day, should be sufficient to increase the choroidal blood flow. Although we did not measure the foveal choroidal blood flow, an improvement of choroidal blood flow after IU might be suggested. This is important because a decrease in choroidal blood flow has been reported to be present in some patients with RP and improving the choroidal circulation could help preserve the cone photoreceptors [26][27][28][29]. This clinical study was conducted by less than 40 patients in each arm in a 24-month observation. To confirm the long-term efficacy and safety of the drug, the phase III clinical trial is now planned as a long-term observation with a large number of patients.