Takahiro Akaishi

Ocular Hypotensive Effects of Anti-Glaucoma Agents in Mice

PURPOSE To evaluate the ocular hypotensive effects induced by topical application of anti-glaucoma agents in mice. METHODS Representative drugs (latanoprost and tafluprost [for prostanoid FP receptor agonists], timolol [for beta-adrenoceptor antagonists], dipivefrin [for alphabeta-adrenoceptor agonists], dorzolamide [for carbonic anhydrase inhibitors], pilocarpine [for muscarinic receptor agonists], bunazosin [for alpha(1)-adrenoceptor antagonists], or brimonidine [for alpha(2)-adrenoceptor agonists]) were used as anti-glaucoma agents; each one being topically applied once in a given male ddY mouse. Intraocular pressure (IOP) was measured using the microneedle method under general anesthesia. IOP was measured before, and at 1, 2, 3, and 4 h after administration of each drug. The contralateral eyes were untreated. At the each time point, the induced IOP reduction was evaluated by calculating the difference in IOP between the treated and untreated eyes in one and the same mouse. RESULTS All of the evaluated anti-glaucoma agents reduced IOP in mice. The 2 prostanoid FP receptor agonists, the beta-adrenoceptor antagonist, and the alphabeta-adrenoceptor agonist began significantly to reduce IOP 2 h after their administration, and mostly induced a long-lasting IOP reduction. The alpha(1)-adrenoceptor antagonist, the alpha(2)-adrenoceptor agonist, the muscarinic receptor agonist, and the carbonic anhydrase inhibitor began reducing the IOP within 1 h after their administration, but their effects waned fairly quickly (the IOP reductions being lost by 3 h after their administration). Concomitant administration of timolol and tafluprost or of dorzolamide and tafluprost induced a significantly greater IOP reduction than that induced by either of the individual components. CONCLUSIONS In this study, all the anti-glaucoma agents tested had apparent ocular hypotensive effects in mice. Our data suggest that the mouse may be a useful animal for the evaluation of the pharmacological effects of agents with various anti-glaucoma mechanisms, and for the evaluation of the enhanced ocular hypotensive effects that may be induced by the concomitant use of 2 anti-glaucoma agents.

Effects of Repeated Administrations of Tafluprost, Latanoprost, and Travoprost on Optic Nerve Head Blood Flow in Conscious Normal Rabbits

PURPOSE The aim of this study was to evaluate and compare the effects of repeated administrations of three prostaglandin F(2alpha) analogs (tafluprost, latanoprost, and travoprost) on optic nerve head (ONH) blood flow in normal rabbits. METHODS Male Dutch rabbits were housed under a 12-h light-dark cycle for use in this study. A quantitative index of blood flow, the squared blur rate (SBR), was determined using laser speckle flowgraphy. Saline, 0.0015% tafluprost, 0.005% latanoprost, or 0.004% travoprost (each 50 microL) was topically administered into the left eye once daily for 28 days. ONH blood flow was measured before the start at the course of treatment (baseline), and on day 14 and/or day 28 [measurements being made at 0, 30, and/or 60 min after drugs application on day 14 and/or day 28]. Heart rate was also measured at these time points. RESULTS Tafluprost, latanoprost, and travoprost each increased the ONH blood flow at all measurement points on day 14 and/or day 28. The 0 min SBR value on day 14 was greater than the baseline SBR value by 8.7% + or - 4.4% for tafluprost and by 5.8% + or - 1.7% for latanoprost. The 0 min SBR value on day 28 was greater than the baseline SBR value by 11.9% + or - 3.9% for tafluprost, by 7.2% + or - 4.3% for latanoprost, and by 6.7% + or - 3.5% for travoprost. The increasing state of the ONH blood flow continued within 60 min after a topical administration of tafluprost, latanoprost, or travoprost. Tafluprost, latanoprost, and travoprost did not change heart rate (vs. the baseline value) at any measurement points. CONCLUSIONS Repeated topical administration of any of the three prostaglandin F(2alpha) analogs increased ONH blood flow in rabbits, without changing heart rate. The increase in ONH blood flow induced by tafluprost was greater than that induced by latanoprost (P = 0.086) or travoprost (P = 0.037) at 60 min on day 28.

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

Purpose:To clarify the mechanism by which bunazosin hydrochloride (BZ), a selective &agr;1-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 10−7 to 10−5 M did not increase MMP-2, -3, and -9 activities in the culture medium of ciliary muscle cells, BZ at 10−7 to 10−5 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.

Metabolism and Ocular Tissue Distribution of an Antiglaucoma Prostanoid, Tafluprost, After Ocular Instillation to Monkeys

PURPOSE To investigate the metabolism of a new antiglaucoma difluoroprostaglandin, tafluprost, in ocular tissues and evaluate the distribution of the parent drug and its metabolites in ocular and systemic tissues after a single ocular administration to cynomolgus monkeys (Macaca fascicularis). METHODS A single dose of an ophthalmic solution containing 0.0005%, 0.005%, or 0.05% [(3)H]tafluprost was topically instilled (20 μL/eye) to male and/or female cynomolgus monkeys to study tissue distribution and metabolism. Blood, ocular/systemic tissues, or excreta were collected until 24 h after dosing. The radioactivity of each sample was measured by liquid scintillation counting, and metabolites were characterized by liquid chromatography-mass spectrometry. The major metabolites found in ocular tissues were intracameraly administered to monkeys to confirm their effect on intraocular pressure (IOP). RESULTS Soon after dosing, high concentrations of drug-related radioactivity were observed in the cornea and bulbar/palpebral conjunctiva, followed by the iris, sclera, choroid with retinal pigmented epithelium, and aqueous humor. The highest concentration of radioactivity concentrations occurred in the anterior and posterior ocular tissues within 2 h after dosing. The radioactivity measured in the plasma and ocular tissues was proportional to the dose administered. The major metabolites of tafluprost identified in the ocular tissues were tafluprost acid and 1,2-dinor- and 1,2,3,4-tetaranor-tafluprost acid. The estimated concentration of tafluprost acid in the aqueous humor and ciliary body was enough to stimulate prostanoid FP-receptors. After hydrolysis to the acid form, the primary metabolic pathway of tafluprost was via β-oxidation and, subsequently, oxidation. No metabolic reactions to the 15-carbon position were observed. Tafluprost acid was shown to significantly lower the IOP, whereas 1,2-dinor- and 1,2,3,4-tetaranor-tafluprost acid did not. CONCLUSIONS Topically administered [(3)H]tafluprost was well absorbed into the ocular and systemic tissues of the primary nonclinical species, monkey. The amount of the pharmacologically active form, that is, tafluprost acid, was high enough to occupy the target FP receptors at the site of action. The pharmacokinetic and metabolic properties of this difluorinated prostaglandin in primates are believed to result in clinical benefits of a long-term IOP-lowering effect.

Advantages of Efficacy and Safety of Fixed-Dose Tafluprost/Timolol Combination Over Fixed-Dose Latanoprost/Timolol Combination

Purpose To compare the safety and efficacy of fixed-dose tafluprost/timolol combination (Taf/T-FDC) with those of fixed-dose latanoprost/timolol combination (Lat/T-FDC) by measuring the intraocular pressure (IOP)-lowering effect, ocular pharmacokinetics, and ocular surface toxicity. Methods The IOP-lowering effect of Taf/T-FDC and Lat/T-FDC in ocular normotensive monkeys was evaluated at 4 and 8 h after instillation in study A, at 12, 14, 16, and 18 h after instillation in study B, and at 24, 26, 28, and 30 h after instillation in study C. Drug penetration into the eye was evaluated by measuring the concentrations of timolol, tafluprost acid (active metabolic form of tafluprost), and latanoprost acid (active metabolic form of latanoprost) using liquid chromatography coupled with tandem mass spectrometry after single instillation of Taf/T-FDC or Lat/T-FDC to Sprague Dawley rats. Cytotoxicity following 1–30 min exposure of SV40-transformed human corneal epithelial cells to Taf/T-FDC or Lat/T-FDC was analyzed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays. Undiluted and 10-fold diluted solutions of each FDC were evaluated. Results The IOP-lowering effect of Taf/T-FDC was almost equivalent to that of Lat/T-FDC at 4–8 h after instillation. The peak IOP reduction of Taf/T-FDC and Lat/T-FDC was observed at 8 h after instillation, and there is no difference between the two. The difference between them was observed at 24–30 h after instillation, and Taf/T-FDC demonstrated a significantly greater IOP-lowering effect than Lat/T-FDC at 24–30 h after instillation. The IOP-lowering effect of Taf/T-FDC was sustained up to 30 h after instillation, while that of Lat/T-FDC had almost disappeared at 28 h after instillation. Timolol concentrations in aqueous humor after Taf/T-FDC instillation were higher than those after Lat/T-FDC instillation (Cmax, 3870 ng/mL vs 1330 ng/mL; AUCinf, 3970 ng·h/mL vs 1250 ng·h/mL). The concentrations of tafluprost acid and latanoprost acid in aqueous humor after instillation of Taf/T-FDC and Lat/T-FDC, respectively, were similar to those after instillation of mono-preparations of tafluprost and latanoprost, respectively. The cytotoxic effect of Taf/T-FDC to the human corneal epithelial cells was significantly lower than that of Lat/T-FDC at all evaluated time points in both undiluted and 10-fold diluted FDCs. Conclusion Taf/T-FDC provides increased IOP-lowering effect duration and lower potential ocular surface toxicity than Lat/T-FDC.

Benefits of Tafluprost and Timolol Fixed-Dose Combination for the Treatment of Glaucoma Are Confirmed by Studies on Experimental Animal Models

PURPOSE To assess the usefulness of 0.0015% tafluprost and 0.5% timolol fixed-dose combination (TT-FDC) for glaucoma, the ocular hypotensive effect of TT-FDC and concentration of tafluprost and timolol in the aqueous humor were compared with those of the concomitant administration of 0.0015% tafluprost and 0.5% timolol with or without an appropriate administration interval. METHODS The ocular hypotensive effect was assessed by intraocular pressure (IOP) measurement in cynomolgus monkeys. Drug penetration into the aqueous humor was estimated by the concentrations of tafluprost acid (active metabolic form of tafluprost) and timolol, which were measured using liquid chromatography-tandem mass spectrometry after administration of tafluprost and timolol to Sprague Dawley rats. RESULTS The ocular hypotensive effect of TT-FDC was equivalent to that of the concomitant administration of timolol and tafluprost at a more than 5-min interval in monkeys. However, the ocular hypotensive effect of the concomitant administration of timolol and tafluprost without an interval (-2.8 ± 0.2 mmHg at peak IOP reduction) was significantly weaker compared with TT-FDC (-4.3 ± 0.5 mmHg at peak IOP reduction, P = 0.008 vs. concomitant administration of timolol and tafluprost) in monkeys. The aqueous humor concentration of the second administered drug (tafluprost) was not affected by the dosing conditions, whereas the concentration of the first instilled drug (timolol) without the interval was lower than that with a 5-min interval (1,200 ng · h/mL vs. 1,890 ng · h/mL in AUC0-4) in rats. CONCLUSION TT-FDC demonstrates a clear benefit by preventing efficacy loss without an appropriate interval in experimental animal models.

Effects of Ocular Hypotensive Agents on Circadian Rhythms of Intraocular Pressure in Rabbits as Measured by Telemetry System

PurposeTo 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 MethodsWe 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 hypo-tensive agents was measured by telemetry during the light phase and the dark phase.ResultsThe values measured by the telemetry system were positively correlated to the value of the anterior chamber pressure measured by a transducer in a 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.ConclusionThese findings indicate that the different effects of ocular hypotensive agents on circadian rhythms of IOP can be measured by a telemetry system. Telemetry may be useful for evaluation of ocular hypotensive agents and the circadian rhythm of IOP. Nippon Ganka Gakkai Zasshi (J Jpn Ophthalmol Soc 107:513–518, 2003)