Effect of Diquafosol Ophthalmic Solution on Airflow-Induced Ocular Surface Disorder in Diabetic Rats.

PURPOSE: To examine the effect of 3% diquafosol ophthalmic solution (DQS) on ocular surface disorders in diabetic model rats maintained in a continuous airflow condition. METHODS: Goto-Kakizaki (GK) rats, a spontaneous model of type 2 diabetes, were exposed to constant airflow for 8 weeks. After the establishment of the animal model in this environment, DQS or saline was instilled six times a day into GK rat eyes for 6 weeks. Schirmer's test was performed before and after 6-week instillations. Corneal fluorescein staining was scored at 2-, 4-, and 6-week instillations. Touch thresholds for the cornea were also determined using a Cochet-Bonnet esthesiometer before and after 6-week instillations. RESULTS: The mean Schirmer's test score after instillation of DQS was twice higher than that recorded for saline alone. DQS significantly decreased corneal staining scores at 4- and 6-week instillations. No changes in touch thresholds were observed before and after 6-week instillations. CONCLUSION: These results suggest that DQS improves corneal epithelial damage by stimulating tear secretion without influencing corneal sensation in diabetic keratopathy. Thus, DQS may have potential for treatment of diabetic patients with dry eye.

Effects of the Selective EP2 Receptor Agonist Omidenepag on Adipocyte Differentiation in 3T3-L1 Cells.

Purpose: We aimed at comparing the effects of omidenepag (OMD) with those of prostaglandin F (FP) receptor agonists (FP agonists) on adipogenesis in mouse 3T3-L1 cells. Methods: To evaluate the agonistic activities of OMD against the mouse EP2 (mEP2) receptor, we determined cAMP contents in mEP2 receptor-expressing CHO cells by using radioimmunoassays. Overall, 3T3-L1 cells were cultured in differentiation medium for 10 days and adipocyte differentiation was assessed according to Oil Red O-stained cell areas. Changes in expression levels of the adipogenic transcription factors Pparg, Cebpa, and Cebpb were determined by using real-time polymerase chain reaction (PCR). OMD at 0.1, 1, 10, and 40 µmol/L, latanoprost free acid (LAT-A) at 0.1 µmol/L, or prostaglandin F2α (PGF2α), at 0.1 µmol/L were added to cell culture media during adipogenesis. Oil Red O-stained areas and expression patterns of transcription factor targets of OMD or FP agonists were compared with those of untreated controls. Results: The 50% effective concentration (EC50) of OMD against the mEP2 receptor was 3.9 nmol/L. Accumulations of Oil Red O-stained lipid droplets were observed inside control cells on day 10. LAT-A and PGF2α significantly inhibited the accumulation of lipid droplets; however, OMD had no effect on this process even at concentrations up to 40 µmol/L. LAT-A and PGF2α significantly suppressed Pparg, Cebpa, and Cebpb gene expression levels during adipocyte differentiation. Conversely, OMD had no obvious effects on the expression levels of these genes. Conclusions: A selective EP2 receptor agonist, OMD, did not affect the adipocyte differentiation in 3T3-L1 cells, whereas FP agonists significantly inhibited this process.

MALDI imaging mass spectrometry revealed atropine distribution in the ocular tissues and its transit from anterior to posterior regions in the whole-eye of rabbit after topical administration.

It is essential to elucidate drug distribution in the ocular tissues and drug transit in the eye for ophthalmic pharmaceutical manufacturers. Atropine is a reversible muscarinic receptor used to treat various diseases. However, its distribution in ocular tissues is still incompletely understood. Matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) evaluates drug distribution in biological samples. However, there have been few investigations of drug distribution in ocular tissues, including whole-eye segments. In the present study, we explored the spatial distribution of atropine in the whole-eye segment by MALDI-IMS, and then evaluated the changes in atropine level along the anterior-posterior and superior-inferior axes. A 1% atropine solution was administered to a rabbit and after 30 min, its eye was enucleated, sectioned, and analyzed by MALDI-IMS. Atropine accumulated primarily in the tear menisci but was found at substantially lower concentrations in the tissue surrounding the conjunctival sacs. Relative differences in atropine levels between the anterior and posterior regions provided insights into the post-instillation behavior of atropine. Atropine signal intensities differed among corneal layers and between the superior and inferior eyeball regions. Differences in signal intensity among tissues indicated that the drug migrated to the posterior regions via a periocular-scleral route. Line scan analysis elucidated atropine transit from the anterior to the posterior region. This information is useful for atropine delivery in the ocular tissues and indicates that MALDI-IMS is effective for revealing drug distribution in whole-eye sections.

A Combination Therapy Targeting Endoglin and VEGF-A Prevents Subretinal Fibro-Neovascularization Caused by Induced Müller Cell Disruption.

Purpose: Subretinal fibroneovascularization is one of the most common causes of vision loss in neovascular AMD (nAMD). Anti-VEGF therapy effectively inhibits vascular leak and neovascularization but has little effect on fibrosis. This study aimed to identify a combination therapy to concurrently inhibit subretinal neovascularization and prevent fibrosis. Methods: We generated transgenic mice in which induced disruption of Müller cells leads to subretinal neovascularization, which is reliably accompanied by subretinal fibrosis. We conducted Western blots and immunohistochemistry to study changes in transforming growth factor-ß (TGFß) signaling including endoglin, a coreceptor essential for TGFß signaling, and then tested the effects of monthly intravitreal injection of anti-VEGF-A and anti-endoglin, either alone or in combination, on the development of subretinal fibroneovascularization in our transgenic mice. Results: Müller cell disruption increased expression of TGFß1, TGFß type 1 receptor, and phosphorylated-Smad3. Endoglin was strongly expressed in subretinal fibroneovascular tissue. Fluorescein angiography and measurements of retinal vascular permeability indicated that intravitreal anti-VEGF-A in combination with anti-endoglin treatment more efficiently inhibited vascular leak compared with either monotherapy. Immunostaining of retinal wholemounts with antibodies against glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1 indicated that the combination therapy also effectively prevented subretinal fibrosis and inhibited microglial activation. Luminex cytokine assays indicated that intravitreal anti-VEGF-A and anti-endoglin treatment, either alone or in combination, reduced the production of IL33 and macrophage inflammatory protein-3α. Conclusions: Our findings offer a potentially novel combination approach to concurrently managing subretinal neovascularization and fibrosis in nAMD.

Effects of a Novel Selective EP2 Receptor Agonist, Omidenepag Isopropyl, on Aqueous Humor Dynamics in Laser-Induced Ocular Hypertensive Monkeys.

PURPOSE: To investigate the mechanism of the intraocular pressure (IOP)-lowering effect of a novel selective prostaglandin E2 receptor 2 (EP2) receptor agonist, omidenepag isopropyl (OMDI). METHODS: The effect of OMDI on IOP and aqueous humor dynamics was evaluated in cynomolgus monkeys with unilateral laser-induced ocular hypertension. In a crossover manner, the hypertensive eye of each monkey was dosed once daily with 20 µL of either 0.002% OMDI or vehicle. On day 7 of dosing, IOP was measured by pneumatonometry, aqueous humor flow and outflow facility were evaluated by fluorophotometry, and uveoscleral outflow was calculated mathematically. Treatments were compared by paired t-tests. RESULTS: OMDI at 0.002% significantly lowered IOP by 27%, 35%, and 44% at 0.5, 1.5, and 4 h after the last dosing, respectively. There was no difference in aqueous humor flow between vehicle and OMDI treatments. When comparing OMDI to the vehicle treatment, outflow facility and uveoscleral outflow were significantly (P < 0.05) increased by 71% and 176%, respectively. CONCLUSIONS: OMDI, a novel IOP-lowering compound, reduced IOP by increasing outflow facility and uveoscleral outflow in nonhuman primates.

Pharmacologic Characterization of Omidenepag Isopropyl, a Novel Selective EP2 Receptor Agonist, as an Ocular Hypotensive Agent.

Purpose: The objective of this study was to investigate the pharmacologic characteristics of omidenepag isopropyl (OMDI), a compound developed as a novel intraocular pressure (IOP)-lowering agent, with better IOP control and fewer side effects than other prostanoid receptor agonists such as prostaglandin F receptor (FP) agonists. Methods: Binding activities of OMDI and its hydrolyzed form, omidenepag (OMD), to human recombinant prostanoid receptors (DP1-2, EP1-4, FP, and IP) were evaluated. Based on these binding assays, the agonistic activities of OMDI and OMD were further evaluated using cultured cells expressing selected prostanoid receptors. The pharmacokinetics of OMDI after topical administration was assessed in rabbits by measurement of the concentrations of both OMDI and OMD in aqueous humor. The ocular hypotensive effect of OMDI was evaluated in ocular normotensive rabbits, dogs, and both ocular normotensive and hypertensive monkeys. Results: OMD was determined to be a selective EP2 receptor agonist. OMDI weakly bound to EP1; however, the agonistic activity of OMDI to this receptor was not demonstrated in the functional assay. After topical administration of OMDI, OMD was detected in aqueous humor whereas OMDI was not detectable. OMDI significantly lowered IOP in both ocular normotensive and hypertensive animals. The significant ocular hypotensive effects of OMDI were demonstrated by both single and repeated dosing, and its effective duration suggests sufficient efficacy by once-daily dosing. Conclusions: These studies demonstrated that OMDI is hydrolyzed in the eye to OMD, an EP2 receptor agonist, with a significant ocular hypotensive effect in both ocular normotensive and hypertensive animal models.

Effects of the new ethacrynic acid oxime derivative SA12590 on intraocular pressure in cats and monkeys.

To evaluate the pharmacological characteristics of SA12590, a new oxime-derivative of the ethacrynic acid (ECA) derivative SA9000, we examined both its ocular hypotensive effects (in ocular normotensive cats and cynomolgus monkeys) and its potential corneal toxicity (in rats). A 50 microl topical administration of 3% SA12590 significantly reduced intraocular pressure (IOP) (by 3.5 mmHg) in anesthetized cats (p<0.05). Twenty-four hours after 3 drops (5-min intervals) of 20 microl 3% SA12590, IOP was reduced by 8 mmHg (p<0.05, n=4) in conscious monkeys without evidence of corneal toxicity. Three days' daily single 20 microl dosing with 3% SA12590 reduced IOP by 4 mmHg (p<0.01, n=3) at 72 h after the first administration in conscious monkeys. The toxicity of topically administered 20 microl 3% SA9000 or SA12590 (3 drops with 5-min intervals) on rat corneal epithelium was assessed using a photo-slit lamp. In this study, 3% SA12590, unlike 3% SA9000, exhibited no corneal toxicity. In a glutathione assay for sulfhydryl (SH) reactivity, SA12590, unlike SA9000, displayed no in vitro SH reactivity. Thus, oxime-modification may both improve efficacy towards IOP upon topical administration and improve the safety profile, probably by enhancing corneal penetration and minimizing SH reactivity-related toxicity. These findings indicate that SA12590 has potential as a new ocular hypotensive drug.

Effects of bunazosin hydrochloride on ciliary muscle constriction and matrix metalloproteinase activities.

PURPOSE: To clarify the mechanism by which bunazosin hydrochloride (BZ), a selective alpha1-adrenoceptor antagonist, increases uveoscleral outflow. METHODS: The effects of BZ on matrix metalloproteinase (MMP) activities in cultured monkey ciliary muscle cells, and on phenylephrine hydrochloride-induced constriction in bovine ciliary muscles, were examined. Also, the possible additive ocular hypotensive effects of BZ and latanoprost (LP) were evaluated in ocular normotensive monkeys. RESULTS: Although BZ at 10 to 10 M did not increase MMP-2, -3, and -9 activities in the culture medium of ciliary muscle cells, BZ at 10 to 10 M inhibited phenylephrine hydrochloride-induced constriction in ciliary muscles. The maximal reduction in intraocular pressure of concomitant administration of BZ and LP was greater than that of BZ alone and tended to be greater than that of LP alone. CONCLUSION: These findings, in normotensive monkeys, indicate that the mechanism whereby BZ increases uveoscleral outflow is independent of an effect on MMPs and is partly due to relaxation of the ciliary muscle. This effect is different from that of LP, which might help to explain the finding that topical concomitant administration of BZ and LP increased AUC(0-6h) value (IOP reduction) to 201% and 145% of BZ and LP given alone, respectively.

New ethacrynic acid derivatives as potent cytoskeletal modulators in trabecular meshwork cells.

A series of ethacrynic acid (ECA) derivatives were synthesized and examined for ocular hypotensive activity. Efficacy was evaluated in a cell-shape assay, using human trabecular meshwork cells, and cytotoxicity in a (3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay, using cultured bovine trabecular meshwork cells. Many of the derivatives demonstrated efficacy equal to or greater than that of ECA. SA9000 was selected as the most promising candidate for a novel ocular hypotensive drug with few side effects.

Pharmacological characteristics of AFP-168 (tafluprost), a new prostanoid FP receptor agonist, as an ocular hypotensive drug.

To evaluate the pharmacological characteristics of AFP-168 (tafluprost), a new prostaglandin (PG) F(2alpha) derivative, we examined its receptor-binding affinities, intraocular pressure (IOP)-lowering effect, effects on aqueous humor dynamics, and stimulating effect on melanogenesis. The receptor-binding profile for AFP-172, a carboxylic acid of AFP-168, was determined by measuring muscle contractions in an organ bath, inhibition of platelet aggregation, and competitive binding of a radio-labelled ligand. For the IOP-measurement study, ocular normotensive and laser-induced ocular hypertensive cynomolgus monkeys were used, and IOP was measured using a pneumatonograph. For the studies of aqueous humor dynamics, IOP (Goldmann applanation tonometry), fluorophotometry, two-level constant pressure perfusion, and isotope dilution and accumulation techniques were used in ocular normotensive monkeys. The melanin contents in the medium and in the cell bodies of cultured B16-F0 melanoma cells were measured. The affinity for the FP receptor shown by AFP-172 (Ki : 0.4 nm) was 12 times that of PhXA85 ( Ki : 4.7 nm), a carboxylic acid of latanoprost. A single application of AFP-168 at 0.0025% significantly lowered IOP in both ocular normotensive and hypertensive monkeys (3.1 and 11.8 mmHg, respectively, p < 0.01) and latanoprost at 0.005% significantly lowered IOP (2.1 mmHg, p < 0.01 and 9.5 mmHg, p = 0.059 respectively). Once daily instillation of AFP-168 at 0.001, 0.0025, or 0.005% for 5 days in normotensive monkeys significantly reduced IOP not only for a few hours, but also at the drug-trough time 24hr after application. Latanoprost at 0.005% also reduced IOP, but not at the drug-trough time. AFP-168 decreased IOP mainly by increasing uveoscleral outflow by 65% (p < 0.05) and, as sometimes seen with other prostanoids, also increased total outflow facility (33% increase, p < 0.05). In cultured B16-F0 melanoma cells, AFP-172 (100 microM) did not stimulate melanogenesis, but PhXA85 (100 microM) did. These findings indicate that AFP-168 has a high affinity for the prostanoid FP receptor, has potent IOP-lowering effects in both ocular normotensive and hypertensive monkeys that exceed those of latanoprost, and has less stimulating effect on melanogenesis in melanoma cells.

New fluoroprostaglandin F(2alpha) derivatives with prostanoid FP-receptor agonistic activity as potent ocular-hypotensive agents.

To find new prostanoid FP-receptor agonists possessing potent ocular-hypotensive effects with minimal side effects, we evaluated the agonistic activities of newly synthesized prostaglandin F(2alpha) derivatives for the prostanoid FP-receptor both in vitro and in vivo. The iris constrictions induced by the derivatives and their effects on melanin content were examined using cat isolated iris sphincters and cultured B16 melanoma cells, respectively. The effects of derivative ester forms on miosis and intraocular pressure (IOP) were evaluated in cats and cynomolgus monkeys, respectively. Of these derivatives, 6 out of 12 compounds were more potent iris constrictors, with EC(50) values of 0.6 to 9.4 nM, than a carboxylic acid of latanoprost (EC(50)=13.6 nM). A carboxylic acid of latanoprost (100 microM) significantly increased the melanin content of cultured B16 melanoma cells, but some 15,15-difluoro derivatives, such as AFP-157 and AFP-172, did not. Topically applied AFP-168, AFP-169 and AFP-175 (isopropyl ester, methyl ester and ethyl ester forms, respectively, of AFP-172) induced miosis in cats more potently than latanoprost. AFP-168 (0.0005%) reduced IOP to the same extent as 0.005% latanoprost (for at least 8 h). These findings indicate that 15,15-difluoroprostaglandin F(2alpha) derivatives, especially AFP-168, have more potent prostanoid FP-receptor agonistic activities than latanoprost. Hence, AFP-168 may be worthy of further evaluation as an ocular-hypotensive agent.

Endogenous glutamatergic synaptic activity elicits acetylcholine release from rat cultured septal cells.

We tested the characteristics of acetylcholine (ACh) release from cultured rat septal cells. The spontaneous release was inhibited by treatment with tetrodotoxin (TTX) and omega-conotoxin (GVIA), indicating that the release was elicited by synaptic activity. The release was also inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor blocker, in both the absence and presence of nerve growth factor (NGF), suggesting that endogenously released glutamate produced the ACh release by stimulating AMPA receptors. This is the first report of detection of the release of ACh by endogenous spontaneous synaptic activity conducted by glutamate AMPA receptor activation in cultured septal cells. This in vitro experimental system is useful for the study of cholinergic functions.

[Effects of ocular hypotensive agents on the circadian rhythm in intraocular pressure in rabbits as measured by telemetry].

PURPOSE: To establish a telemetry system for measuring intraocular pressure (IOP) in rabbits and to evaluate the effects of topical application of ocular hypotensive agents on the circadian rhythm of IOP. SUBJECTS AND METHODS: We developed a telemetry system in rabbits housed under a 12-hour light-dark cycle (light and dark phases: 7:00-19:00, 19:00-7:00, respectively). The IOP resulting from a single topical application of ocular hypotensive agents was measured by telemetry during the light phase and the dark phase. RESULTS: The values measured by the telemetry were positively correlated to the value of the anterior chamber pressure measured by a transducer in range from 5 to 50 mmHg (r = 0.987). A single topical application of timolol maleate (0.5%), dorzolamide hydrochloride (1%), and dipivefrine hydrochloride (0.1%) caused no significant reduction in IOP in the light phase, but they did in the dark phase. A single topical application of bunazosin hydrochloride (0.01% or 0.1%) had significant ocular hypotensive effects in both phases. CONCLUSION: These findings indicate that the different effects of ocular hypotensive agents on circadian rhythms of IOP can be measured by the telemetry. Telemetry may be useful for evaluation of ocular hypotensive agents and the circadian rhythm of IOP.