Ocular Tissue Distribution of Omidenepag Isopropyl in Rabbits and Cynomolgus Monkeys

Abstract

Purpose: To evaluate the ocular distribution of omidenepag isopropyl (OMDI) and its active form omidenepag (OMD), an EP2 receptor agonist, after topical administration of OMDI into rabbit and monkey eyes, and to determine whether OMDI and OMD interact with target receptors or enzymes of other antiglaucoma agents.

Methods: Both eyes of six rabbits and of 14 monkeys were topically instilled with 0.03% [¹⁴C]-OMDI. Rabbits were sacrificed after one to four hours, and ocular tissues were collected. Monkeys were sacrificed after 0.25 to 24 hours, and blood and ocular tissues were collected. Radioactivity was measured in each sample. The interactions of OMDI and OMD with the receptors and enzymes associated with the mechanisms of action of other antiglaucoma agents were evaluated.

Results: Most radioactivity applied to rabbit eyes was recovered as OMD from the cornea, aqueous humor, and iris-ciliary body. Similarly, high concentrations of radioactivity were observed in monkey cornea, bulbar/palpebral conjunctiva, and trabecular meshwork. OMD bound to EP2 receptors, but neither OMD nor OMDI bound to α_2A, β₁, and β₂ adrenergic receptors or inhibited enzymatic activities of CA1 and CA2. OMD and OMDI had little or no effect on ROCK1 and ROCK2.

Conclusions: OMDI rapidly permeates rabbit and monkey corneas and is converted to OMD, which distributes into anterior ocular tissues. Neither OMD nor OMDI interacted with the target receptors or enzymes of other antiglaucoma agents, suggesting that OMD interacts highly selectively with EP2 receptors.

Translational Relevance: OMDI is a specific antiglaucoma agent that interacts selectively with ocular EP2 receptors.

Introduction

Glaucoma, including primary open-angle glaucoma (POAG), and related conditions such as ocular hypertension (OHT) are treated mostly with topical medications that reduce intraocular pressure (IOP).^1–3 Prostaglandin F_2α analogues (PGAs) and prostaglandin F (FP) receptor agonists, such as latanoprost, bimatoprost, travoprost, and tafluprost, are the most commonly used front-line antiglaucoma drugs.^1,4 Other agents used to treat glaucoma include β-blockers, α₂-adrenergic receptor agonists, carbonic anhydrase inhibitors, parasympathomimetics, nitric oxide donating prostaglandins, and Rho kinase inhibitors.^1,5–7 Although these drug classes having relatively good safety and efficacy profiles, they also have side effects, with fatigue being associated with β-blockers and α₂ agonists, depression with β-blockers, and periorbitopathy with PGAs/FP agonists.^1,8 

Prostaglandin E₂ receptors of the EP2 subtype (EP2 receptors) are distributed throughout ocular tissues, including the iris, ciliary body, and sclera.⁹ Omidenepag (OMD) is a novel non-prostaglandin molecule that binds strongly (Ki 3.6 nM) and selectively to EP2 receptors, but weakly to other prostanoid receptors (Ki >6000 nM).⁹ OMD is administered as a prodrug, omidenepag isopropyl (OMDI), which is hydrolyzed in the eye (largely in the cornea) to its active form.^9–11 The binding of OMD to EP2 receptors upregulates the expression of matrix metalloproteinases and downregulates the expression of collagen types XII and XIII, widening intramuscular spaces and improving the drainage of aqueous humor.^12–15 Unlike PGAs, OMD does not bind to FP receptors,⁹ eliminating or greatly reducing the risk of side effects like eyelash growth and pigmentation in periorbitopathy.^1,8,16,17 

Topically administered OMDI has been found to significantly reduce IOP in ocular normotensive rabbits, dogs, and monkeys and in monkeys with laser-induced ocular hypertension.⁹ These reductions were due primarily to changes in the architecture of tissues belonging to both the uveoscleral and trabecular outflow pathways, improving the drainage of aqueous humor.¹² Moreover, the combination of OMDI and other IOP-lowering drugs like timolol resulted in greater reductions in IOP than either agent alone.¹⁸ 

In addition to significantly reducing IOP in animals,⁹ clinical trials have shown that OMDI significantly reduced IOP in patients with POAG and OHT. Dose finding studies showed that the optimal concentration of OMDI was 0.002%¹⁹ and that once-daily dosing was associated with significantly lower rates of side effects than twice-daily dosing.²⁰ The phase 3 AYAME trial found that treatment with 0.002% OMDI was non-inferior to treatment with 0.005% latanoprost for four weeks.²¹ In addition, the phase 3 FUJI and Spectrum-5 trials found that treatment with OMDI for four weeks and three months, respectively, was effective in patients with poor or no response to latanoprost.^5,22 Moreover, the phase 3 RENGE trial found that OMDI was effective for 12 months in groups of patients with baseline IOPs of 16 to 22 mm Hg and 22 to 34 mm Hg.²³ 

This study sought to evaluate the ocular tissue distribution and metabolite profile of OMDI in animal eyes after topical administration, and to determine whether OMDI and OMD interact with the target molecules of other antiglaucoma agents. In one series of experiments, the metabolite profile of OMDI was assessed following the topical administration of [¹⁴C]-OMDI ophthalmic solution into rabbit eyes. In the second series of experiments, the ocular tissue distribution of radioactivity was assessed following topical administration of [¹⁴C]-OMDI into the eyes of cynomolgus monkeys. In the last series of experiments, the interactions of OMD and OMDI with the target receptors or enzymes of previously identified antiglaucoma agents were assessed using receptor binding and enzymatic assays. 

Material and Methods

Chemicals

OMDI and its active metabolite OMD were supplied by UBE Corporation (Yamaguchi, Japan). The [¹⁴C]-OMDI was synthesized by Sekisui Medical Co., Ltd. (Tokyo, Japan) and supplied at a specific activity of 4.33 MBq/mg. The [¹⁴C]-OMDI was dissolved in borate buffer containing polyoxyl 35 castor oil, EDTA (disodium salt), glycerin, benzalkonium chloride, and sorbic acid, yielding final ophthalmic solutions of 0.03% [¹⁴C]-OMDI. 

Animals

Rabbits

Male Japanese White rabbits, aged eight to nine weeks and weighing 1.6 to 1.8 kg, were obtained from Kitayama Labes Co., Ltd. (Nagano, Japan). The rabbits, which had not been used in any previous research study, were maintained individually in cages under controlled conditions and fed water and a commercially available diet as desired. All rabbit experimental procedures were performed in compliance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research, and were approved by the Animal Care and Use Committee of Santen Pharmaceutical Co., Ltd. 

Monkeys

Male cynomolgus monkeys (Macaca fascicularis), aged two to four years and weighing 2.5 to 3.8 kg, were maintained individually in cages under controlled conditions and fed water and a commercially available diet as desired. All experimental procedures in monkeys were in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care and Use Committees of Santen Pharmaceutical Co., Ltd. and of the contract laboratories. 

Ocular Tissue Distribution of [¹⁴C]-OMDI

The [¹⁴C]-OMDI was administered topically to both eyes of each rabbit by instilling 50 µL of 0.03% [¹⁴C]-OMDI solution (15 µg/eye) onto the cornea of each eye using a micropipette. Two rabbits each were sacrificed after one, two, and four hours by injection of an overdose of sodium pentobarbital solution into the marginal ear vein. The eyeballs were enucleated, washed with saline solution, and kept on ice. The aqueous humor of each eye was immediately collected with a syringe. The cornea and iris-ciliary body of each eye were immediately removed, rinsed with saline solution, blotted dry, and weighed. These samples were kept on ice until processed. 

The [¹⁴C]-OMDI was administered topically to both eyes of each monkey by instilling 30 µL of 0.03% [¹⁴C]-OMDI solution (9 µg/eye) onto the cornea of each eye using a micropipette. Blood samples (2–5 mL) were collected from the femoral vein of each monkey into tubes containing sodium heparin anticoagulant immediately prior to sacrifice, and aliquots of blood samples were centrifuged to obtain plasma. Two monkeys each were sacrificed after 0.25, one, two, four, eight, 12, and 24 hours by injection of an overdose of sodium pentobarbital solution. Both eyes of each animal were enucleated and rinsed with saline solution, and samples of aqueous humor and bulbar and palpebral conjunctiva were collected. Each eye was subsequently frozen in liquid nitrogen and kept on dry ice, and all other ocular tissues (choroid including retinal pigmented epithelium [RPE], ciliary body, cornea, iris, lens, retina, sclera, trabecular meshwork, and vitreous humor) were collected. Each sample, except for vitreous humor, was rinsed with saline solution, blotted dry, and weighed. 

Measurement of Radioactivity

Rabbits

Radioactivity in each sample obtained from rabbit eyes was measured using a Tri-Carb 4910TR liquid scintillation counter (LSC; PerkinElmer Inc., Waltham, MA, USA) and Ultima Gold scintillation cocktail (PerkinElmer Inc.). The radioactive components of ocular tissue samples were separated using a HP1100 HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA), with a FLO-ONE 525TR flow scintillation analyzer (PerkinElmer Inc.) and a Sunfire C18 analytical column, 4.6 mm I.D. × 100 mm, particle size 3.5 µm (Waters Corporation, Milford, MA, USA). Aliquots (100 µL) of aqueous humor were immediately mixed with 200 µL acetonitrile. Cornea and iris-ciliary body samples were immediately homogenized in 500 µL acetonitrile/water (1:1) using a cell destructive device (ShakeMaster Auto, Biomedical Science Ltd., Tokyo, Japan). 

A 50 µL aliquot of each mixture of aqueous humor and acetonitrile was dissolved in 1 mL Soluene-350 (PerkinElmer Inc.) and mixed with 10 mL of Ultima Gold, and radioactivity was measured by LSC. The remainder of each solution was spun in a centrifuge and decanted, and the supernatant was analyzed by HPLC. A 50 µL aliquot of each supernatant was mixed with 10 mL of Ultima Gold and radioactivity was measured by LSC. In addition, each 100 µL aliquot of cornea and iris-ciliary body homogenates was weighed and dissolved in 1 mL Soluene-350, with each subsequently mixed with 10 mL Ultima Gold for LSC analysis. The remaining homogenate was centrifuged and decanted, and the supernatant was analyzed by HPLC. A 100 µL aliquot of each supernatant was weighed, mixed with 10 mL of Ultima Gold, and analyzed by LSC. 

Monkeys

Monkey tissue samples were combusted in a Model 307 Sample Oxidizer (PerkinElmer Inc.), with the resulting [¹⁴C]-CO₂ trapped in a mixture of Perma Fluor (PerkinElmer Inc.) and Carbo-Sorb (PerkinElmer Inc.). Radioactivity in each sample was measured using a Model 2900TR LSC (PerkinElmer Inc.) and Ultima Gold XR scintillation cocktail (PerkinElmer Inc.). Blood samples were placed in combustion cones, followed by combustion and analysis of radioactivity by LSC. Plasma, aqueous humor, and vitreous humor samples were directly analyzed by LSC. Conjunctiva (bulbar and palpebral), cornea, lens, sclera, and trabecular meshwork tissue samples were digested in NaOH and analyzed by LSC. Choroid-RPE, ciliary body, iris, and retina samples were digested in NaOH, neutralized with HCl, combusted, and analyzed by LSC. 

Receptor Binding Assays

Assays measuring the binding of OMD to EP2 receptor were performed by Sekisui Medical Co., Ltd. (Tokyo, Japan), and OMD and OMDI binding assays were performed by Eurofins Panlabs Discovery Services Taiwan, Ltd. (New Taipei City, Taiwan), as previously described.^24–27 The binding affinities of 10 µM OMD and 10 µM OMDI to α_2A, β₁, and β₂ adrenergic receptors were determined by measuring the inhibition by unlabeled compounds of the binding of radiolabeled ligands to each receptor. All assays performed by Sekisui Medical Co., Ltd. and Eurofins Panlabs Discovery Services Taiwan, Ltd., included appropriate positive control samples to validate the performance of each assay system. 

Enzyme Assays

The abilities of 10 µM OMD and 10 µM OMDI to inhibit the enzymatic activities of rho-associated coiled-coil containing protein kinases 1 (ROCK1) and 2 (ROCK2) and carbonic anhydrases 1 (CA1) and 2 (CA2) were quantified by biochemical enzymatic assays performed by Eurofins Panlabs Discovery Services Taiwan, Ltd., as described.^28–30 Briefly, human ROCK1 (catalytic domain, amino acids 17-535 of accession number NP_005397.1) and ROCK2 (catalytic domain, amino acids 11-552 of accession number NP_004841.2) were expressed in insect Sf21 cells. A synthetic peptide, long S6 kinase substrate (amino acid sequence: KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK), and [γ³²P]-ATP were incubated with ROCK1 and ROCK2 for 30 minutes at 37°C, and their enzymatic activities were measured by determining the amount of [³²P]-long S6 kinase substrate formed. 

The abilities of 10 µM OMD and 10 µM OMDI to inhibit the enzymatic activities of ROCK1 and ROCK2 were also determined by off-chip mobility shift assays performed by Carna Biosciences, Inc. (Kobe, Japan).³¹ Human ROCK1 (catalytic domain, amino acids 1-477 of accession number NP_005397.1) and ROCK2 (catalytic domain, amino acids 1-553 of accession number NP_004841.2) were expressed as N-terminal GST-fusion proteins of 82 kDa and 91 kDa, respectively, using a baculovirus expression system. GST-ROCK1 and GST-ROCK2 were purified by glutathione sepharose chromatography. A synthetic peptide, LIMKtide (amino acid sequence: KPDRKKRYTVVGNPY), was incubated with ROCK1 and ROCK2 for one hour at room temperature, and each reaction mixture was applied to a LabChip system (PerkinElmer Inc.) to separate and quantify the product and substrate peptide peaks. All assays performed by Eurofins Panlabs Discovery Services Taiwan, Ltd. and Carna Biosciences, Inc. included appropriate positive controls to validate the performance of each assay system. 

Data Analysis

The maximum concentration (C_max) of radioactivity in blood, plasma, and ocular tissues and the time to reach maximum concentration (T_max) were determined by visual inspection of the raw data. Pharmacokinetic parameters calculated in monkeys included the elimination half-life (t_1/2) and the areas under the concentration-time curves from time 0 to the last measurable time point (AUC_0-t) and from time 0 to infinity (AUC_0-∞). Pharmacokinetic parameters were calculated using WinNonlin Professional Edition software, Version 5.2. 

Results

Rabbit Study

Figure 1 and Table 1 show the mean concentrations of radioactivity in the cornea, aqueous humor, and iris-ciliary body of each rabbit eye one, two, and four hours after topical administration of a single dose of 0.03% [¹⁴C]-OMDI. Evaluations of the recovery of radioactivity showed that 59.5% to 63.6% of radioactivity applied was recovered from the cornea, 93.6% to 99.5% from the aqueous humor, and 61.4% to 68.0% from the iris-ciliary body. The concentrations of total radioactivity in the cornea reached C_max at one hour, the first sampling point, and declined thereafter. The concentrations of OMDI in the cornea were below the lower limit of quantification at one, two, and four hours after topical administration. Except for the unextracted fraction, most of the radioactivity was associated with OMD. OMD was the major metabolite in the cornea, with a maximum concentration of 4130 ng eq/g (7930 nM) at one hour after topical administration. The concentrations of total radioactivity in the aqueous humor and iris-ciliary body reached C_max at two hours and one hour, respectively, declining thereafter. The concentrations of OMDI in the aqueous humor and iris-ciliary body were below the lower limits of quantification. Most of the radioactivity in aqueous humor and iris-ciliary body was associated with OMD, although other metabolite peaks were detected in aqueous humor. OMD was the major metabolite in aqueous humor and iris-ciliary body, with maximum concentrations of 141 ng eq/mL (271 nM) at two hours and 84.9 ng eq/g (163 nM) at one hour after topical administration, respectively. 

Figure 1.

 

Mean concentrations of radioactivity in the (A) cornea, (B) aqueous humor, and (C) iris-ciliary body one, two, and four hours after topical administration of 0.03% [¹⁴C] omidenepag isopropyl into the eyes of male Kbs:JW rabbits. Each point represents the mean ± SD of radioactivity in tissue samples from four eyes (two rabbits). Omidenepag isopropyl concentrations are not shown because they were below the lower limit of quantification in two or more eyes at each time point.

Table 1.

 

Mean Concentrations of Radiolabeled Omidenepag Isopropyl, Omidenepag, and Other Metabolites in the Cornea, Aqueous Humor, and Iris-Ciliary Body After a Single Topical Administration of 0.03% [¹⁴C]-Omidenepag Isopropyl Into the Eyes of Male Albino Rabbits (15 µg/Eye)

 

Monkey Study

After topical administration of a single dose of 0.03% [¹⁴C]-OMDI into both eyes of male cynomolgus monkeys, the T_max of radioactivity in blood and plasma was observed at 0.25 hours, the first sampling point (Fig. 2, Table 2). These concentrations, however, subsequently declined and were below the lower limit of quantification after two and four hours, respectively. High concentrations of [¹⁴C]-OMDI-related radioactivity were observed in the cornea, bulbar/palpebral conjunctiva, and trabecular meshwork soon after administration, followed by the detection of radioactivity in the sclera, iris, aqueous humor, ciliary body, and choroid-RPE. 

Figure 2.

 

Mean concentrations over time of radioactivity in blood, plasma, and ocular tissues after a single topical administration of 0.03% [¹⁴C]-omidenepag into both eyes of male cynomolgus monkeys (9 µg/eye). Each value represents the mean of four eyes of two monkeys.

Table 2.

 

Mean Concentrations of Radioactivity in Blood, Plasma and Ocular Tissues After a Single Topical Administration of 0.03% [¹⁴C]-Omidenepag Isopropyl Into Both Eyes of Male Cynomolgus Monkeys (9 µg/Eye)

 

The C_max of radioactivity in the cornea (1250 ng eq/g), bulbar conjunctiva (541 ng eq/g), palpebral conjunctiva (139 ng eq/g), and trabecular meshwork (467 ng eq/g) occurred 15 minutes after dosing. Peak concentrations of radioactivity were observed at one hour in the sclera (54.3 ng eq/g) and iris (29.0 ng eq/g), at two hours in the choroid-RPE (8.40 ng eq/g), and at four hours in the aqueous humor (22.1 ng eq/g) and ciliary body (14.0 ng eq/g). Quantifiable concentrations of radioactivity were observed after 24 hours only in the cornea, bulbar conjunctiva, palpebral conjunctiva, trabecular meshwork, sclera, and aqueous humor. The concentrations of radioactivity in the vitreous humor, lens, and retina were below the lower limits of quantification throughout the entire sampling period. 

The pharmacokinetic profiles of radioactivity were similar and biphasic in the cornea, conjunctiva (bulbar and palpebral), and trabecular meshwork (Fig. 2). The initial phase of radioactivity in these four tissues appeared up to two hours after topical administration, followed by a terminal elimination phase that continued throughout the time course of the study. The terminal elimination phase for the sclera was parallel to that for the bulbar conjunctiva. Evaluation of the pharmacokinetic parameters of radioactivity showed that the t_1/2 for the cornea (4.52 hours) was 1.44- to 2.35-fold lower than that for the other tissues, but similar to that for aqueous humor (3.99 hours) (Table 3). Estimated exposure (AUC_0-∞) was highest in the cornea and bulbar conjunctiva, followed by the trabecular meshwork, palpebral conjunctiva, and sclera. The cornea exhibited the highest AUC_0-∞, which was more than twofold greater than that of the bulbar conjunctiva and trabecular meshwork. The AUC_0-∞ values for the aqueous humor, palpebral conjunctiva, and sclera were seven- to 40-fold lower than the AUC_0-∞ for the cornea. Moreover, the choroid-RPE, ciliary body, and iris were exposed to minimal levels of radioactivity. 

Table 3.

 

Pharmacokinetic Parameters for Radioactivity in Blood, Plasma, and Ocular Tissues After a Single Topical Administration of 0.03% [¹⁴C]-Omidenepag Isopropyl Into Both Eyes of Male Cynomolgus Monkeys (9 µg/Eye)

 

Binding Affinities of OMD and OMDI to α_2A, β₁, and β₂ Adrenergic Receptors and EP2 Receptor

To determine whether OMD and OMDI interact with the target receptors of other antiglaucoma agents, such as α₂-agonists (e.g., brimonidine) and β-blockers (e.g., timolol maleate), the abilities of unlabeled 10 µM OMD and OMDI to inhibit the binding of radiolabeled ligands to the α_2A, β₁, and β₂ adrenergic receptors were evaluated (Table 4). Because OMD is an EP2 agonist, EP2 receptor was also evaluated. Unlabeled OMD and OMDI did not inhibit the binding of radiolabeled ligands to any of these receptors, except for EP2 receptor, indicating that both compounds did not interact with the target receptors of antiglaucoma α₂-agonists and β-blockers. 

Table 4.

 

Inhibitory Effects of OMD and OMDI on α_2A-Adrenergic Receptor, β-Adrenergic Receptors, EP2 Receptor, and the Enzymatic Activities of CA1 and CA2

 

Inhibitory Effects of OMD and OMDI on CA1 and CA2

To evaluate whether OMD and OMDI interact with CA1 and CA2, the target enzymes of other antiglaucoma agents such as brinzolamide and dorzolamide, the abilities of unlabeled 10 µM OMD and OMDI to inhibit the enzymatic activities of CA1 and CA2 were determined. Treatment with 10 µM OMD and 10 µM OMDI did not inhibit the activities of all enzymes tested (Table 4), indicating that OMD and OMDI did not interact with the target enzymes of brinzolamide and dorzolamide. 

Inhibitory Effects of OMD and OMDI on ROCK1 and ROCK2

To evaluate whether OMD and OMDI interact with ROCK1 and ROCK2, the target enzymes of the antiglaucoma agents such as netarsudil and ripasudil, the half maximal inhibitory concentrations (IC₅₀) of OMD and OMDI on ROCK1 and ROCK2 were determined. Preliminary data indicated that treatment with 10 µM OMDI inhibited ROCK1 and ROCK2 activity ≥50% (data not shown). In one set of experiments using a long S6 kinase substrate, the IC₅₀ values of OMD for ROCK1 and ROCK2 were 18,400 and 16,600 nM, respectively.³² In comparison, the present study found that the IC₅₀ values of OMDI for ROCK1 and ROCK2 were 8700 and 6600 nM, respectively (Table 5). In another set of experiments using LIMKtide, OMD and OMDI showed IC₅₀ values ≥30,000 nM, the maximum concentration evaluated, for ROCK1 and ROCK2. These findings indicated that OMD and OMDI showed little or no interaction with the target enzymes of netarsudil and ripasudil, with IC₅₀ values >2000-fold higher for ROCK1 and ROCK2 than for EP2 receptor (8.3 nM).⁹ 

Table 5.

 

Inhibitory effects of OMD and OMDI on ROCK1 and ROCK2

 

Discussion

The present study found that, after its topical administration to rabbit eyes, the prodrug OMDI was rapidly metabolized to its pharmacologically active form OMD in the cornea, and that this active form was absorbed into the aqueous humor and iris-ciliary body. Because the concentration of radioactivity in the unextracted fraction was lower than the concentration of labeled OMD in each sample, the low recovery of radioactivity from cornea and iris-ciliary body was not likely to affect these results. 

Many ocular drugs and prodrugs containing an ester or amide bond are hydrolyzed in the ocular tissues of human and animal species. For example, prostaglandin analogues/FP agonists such as tafluprost, latanoprost, and dipivefrin are hydrolyzed to their active forms by corneal esterases, enabling their use in glaucoma treatment.^33–35 Esterase activities and protein contents have been well characterized in rabbit ocular tissues, with typical substrates of esterases being metabolized.³⁶ These findings suggested that OMDI would be rapidly metabolized to OMD in the cornea, with the active form subsequently absorbed into intraocular tissues. 

Based on the C_max values of OMD in rabbit cornea, aqueous humor and iris-ciliary body after topical administration of 0.03% [¹⁴C]-OMDI, topical application of 0.002% OMDI ophthalmic solution to patients would result in OMD concentrations in the cornea, aqueous humor, and iris ciliary body of 275 ng eq/g (529 nM), 9.4 ng eq/g (18.1 nM), and 5.66 ng eq/g (10.9 nM), respectively. The in vitro EP2 agonistic activity of OMD (8.3 nM)⁹ suggests that topically applied 0.002% OMDI would yield sufficient concentrations of OMD in aqueous humor and iris-ciliary body to stimulate EP2 receptors in vivo. 

Evaluation of the pharmacokinetic properties of OMDI in male cynomolgus monkeys showed that, after topical application of a single dose of 0.03% [¹⁴C]-OMDI to both eyes, radioactivity was present in blood and plasma for up to one and two hours, respectively. Minimal systemic exposure was observed, with no notable partitioning of radioactivity into the cellular component of blood. Radioactivity was widely distributed to anterior ocular tissues, with the highest concentrations observed in the cornea, bulbar conjunctiva, palpebral conjunctiva, and trabecular meshwork 0.25 hours after administration. Moreover, radioactivity remained quantifiable in these tissues for up to 24 hours but could not be detected in the pigmented tissues of the eye, including the choroid-RPE, ciliary body, and iris. These findings suggest that OMDI, OMD, or both have a low affinity for melanin. 

The pharmacokinetic profiles of OMD in the cornea, conjunctiva (bulbar and palpebral), and trabecular meshwork appeared to be biphasic. The t_1/2 values calculated for the cornea and aqueous humor were similar, suggesting that these tissues were in equilibrium during the terminal elimination phase. The cornea and bulbar conjunctiva exhibited the highest estimated exposure, as determined by AUC_0-∞, followed by the trabecular meshwork, palpebral conjunctiva, aqueous humor, and sclera. Taken together, these results suggest that topically applied OMDI initially permeates the cornea and diffuses into the aqueous humor, with exposure being greatest in the dosed areas of the eye and in tissues involved in the trabecular and uveoscleral outflow pathways, which facilitate aqueous humor clearance from the anterior chamber. Tafluprost was reported to be hydrolyzed to tafluprost acid in monkey cornea, aqueous humor, iris, ciliary body and conjunctiva,³⁴ suggesting that, after application of the [¹⁴C]-OMDI, most of the radioactivity in monkey ocular tissues would be in the form of OMD. Throughout the entire sampling period, the concentrations of radioactivity in vitreous humor, lens, and retina were below the lower limits of quantification, suggesting that OMDI and/or OMD were not distributed to these tissues. 

The C_max values of radioactivity in the trabecular meshwork and ciliary body after topical administration of 0.03% [¹⁴C]-OMDI in monkeys, were 467 ng eq/g (897 nM) and 14.0 ng eq/g (26.9 nM), respectively, suggesting that application of 0.002% OMDI ophthalmic solution, the approved concentration in humans, would yield concentrations of radioactivity in trabecular meshwork and ciliary body of 31.1 ng eq/g (59.8 nM) and 0.93 ng eq/g (1.79 nM), respectively.³⁷ Neither OMD nor OMDI interacted with the target receptors or enzymes of other antiglaucoma agents, except for ROCK1 and ROCK2. The enzymatic activities of ROCK1 and ROCK2 were assessed using two substrates with completely different amino acid sequences. The assay using long S6 kinase substrate showed that OMD and OMDI were very weak inhibitors of ROCK1 and ROCK2, with IC₅₀ values >12.5 times higher than the maximum OMD concentration (529 nM) in rabbit cornea following the application of 0.002% OMD ophthalmic solution, a concentration considered sufficiently low to not actually inhibit ROCK1 and ROCK2 in any ocular tissues. The other assay using LIMKtide showed that OMD and OMDI did not inhibit ROCK1 and ROCK2. Although the enzymes used in these two assays did not differ significantly, as their sequences largely overlapped and included the same catalytic domain, the amino acid sequences of the substrates (long S6 kinase substrate and LIMKtide) differed markedly, with no homology. Accordingly, the differences in amino acid sequences of the substrates may have resulted in differences in the steric structures of the enzyme-substrate complexes, affecting the binding of OMD and OMDI to these complexes. Although their mode of inhibition is unknown, OMD and OMDI likely showed weak inhibitory effects in the assay using long S6 kinase substrate by binding to the enzyme-substrate complex. In the assay using LIMKtide, however, OMD and OMDI did not interact with the enzyme-substrate complex. Taken together, these findings suggested that OMD and OMDI did not interact with ROCK1 and ROCK2. Namely, it was indicated that OMD interacts only with EP2 receptor and is known to have an IOP-lowering effect. 

This study had several limitations, including the small numbers of rabbits and monkeys, and the use of only male animals. Moreover, these animals were ocular normotensive, with apparently normal vision, and lacked the risk factors observed in patients with glaucoma, such as diabetes, heart disease, and exposure to smoking, as well as genetic factors. Thus it is not clear whether OMDI or OMD would behave differently in animals and in patients with glaucoma or OHT. However, extrapolation of the current data from animals to pharmacokinetics in humans^38,39 suggests that the mechanism by which OMDI reduces IOP in glaucoma patients may be due to the ocular distribution of OMD following topical administration of OMDI and the estimated drug concentrations in ocular tissues. 

In conclusion, these results demonstrate that OMDI rapidly permeates the corneas of rabbits and monkeys, that OMDI is converted to its active form in rabbit and monkey corneas, and that OMD distributes into anterior ocular tissues, including the aqueous humor, trabecular meshwork and iris-ciliary body. The ability of OMDI to reduce IOP is due primarily to the EP2 receptor agonist activity of OMD, with the contribution of other activities, including the effects of OMD on the target receptors and enzymes of other antiglaucoma agents, being negligible. 

Acknowledgments

Editorial support was provided by BelMed Professional Resources, with funding provided by Santen. 

Supported by Santen Pharmaceutical Co., Ltd. 

Disclosure: K. Yamamura, Santen Pharmaceutical (E); H. Mano, Santen Pharmaceutical (E); M. Fuwa, Santen Pharmaceutical (E); R. Iwamura, UBE Corporation (E); N. Odani-Kawabata, Santen Pharmaceutical (E)