Latanoprost Free Acid Research Articles

Methodology for high-performance liquid chromatography detection of latanoprost and latanoprost free acid

AbstractA gradient high-performance liquid chromatography (HPLC) method has been developed to determine the concentrations of latanoprost (LP) and latanoprost free acid (LPA) in aqueous solutions. It is novel due to a combination of its simplicity, speed, and detection capability in aqueous solutions for both active drug (LPA) and prodrug (LP). This method is applicable for the research and development of novel drug delivery devices and quality control assays for experimental and commercial laboratory settings, as it allows for a high sample throughput. Samples were chromatographed across a C18 + 2.7 µm 4.6 × 7.5 mm reversed-phase column with gradient elution using a mobile phase of aqueous acetic acid (pH 3.1) and acetonitrile with 0.1% acetic acid. UV spectrophotometry was used to monitor the eluents at 210 nm. Drug concentrations from 1.0 to 150 μg mL−1 were tested, with good linearity observed across the range. LPA had a signature peak at approximately 4.82 min (SD < 0.08) and LP at 9.27 min (SD < 0.07). For both drug and pro-drug, LOD and LOQ were 1.0 and 2.5 μg mL−1, respectively. This assay which accurately measures both prodrug and drug in a single injection, has significant applicability in determining the release kinetics of novel LP drug delivery systems.

Effect of Latanoprostene Bunod on the Permeability of Trabecular Meshwork Cells

Purpose: To investigate the effects of latanoprostene bunod (LBN) on nitric oxide (NO) production and permeability in human trabecular meshwork cells (HTMC).Methods: HTMC were treated with 50 and 100 µM LBN and latanoprost free acid (LAT) for 30 minutes. Additionally, 100 µM LBN was co-exposed to 0.5 mM L-NAME (N-Nitroarginine methyl ester). Cellular viability and NO production were measured using MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) and Griess assays. The permeability and resistance of the HTMC monolayer were evaluated by trans-endothelial electrical resistance (TEER) and carboxyfluorescein permeability.Results: Exposure to 100 µM LBN led to increased NO production, whereas co-exposure to L-NAME reduced NO production. Treatment with 100 µM LBN decreased the TEER of the HTMC monolayer. LBN exposure heightened carboxyfluorescein permeability, but co-exposure to 100 µM LBN and L-NAME reduced permeability. LAT treatment did not affect NO production or permeability.Conclusions: LBN increased the permeability of the HTMC monolayer and increased NO production. Therefore, LBN might increase trabecular outflow in addition to promoting uveoscleral outflow.

Effect of Prostaglandin E2 Agonist Omidenepag on the Expression of Matrix Metalloproteinase in Trabecular Meshwork Cells

PurposeTo investigate the effects of prostaglandin E2 agonist omidenepag (OMD) on the expression of matrix metalloproteinase (MMP) in human trabecular meshwork (TM) cells.MethodsPrimarily cultured human TM cells were exposed to 0, 1, 10, or 40 μmol/L OMD for 3 days. The permeability through the TM cell monolayer was assessed using carboxyfluorescein. Expressions of messenger ribonucleic acid and protein levels of MMP-1, MMP-3, and MMP-9 were measured by reverse transcription polymerase chain reaction and Western blotting, respectively. Also, the permeability, expression of messenger ribonucleic acid, and protein levels of MMPs were measured after exposure to 1 μmol/L latanoprost free acid (LAT).ResultsOMD and LAT did not affect the cellular survival (all p > 0.05). Each concentration of OMD and LAT did not affect the permeability of carboxyfluorescein significantly (all p > 0.05). LAT increased the level of MMP-1 protein but did not increase the levels of MMP-3 and MMP-9 proteins. Each concentration of OMD did not affect the levels of MMP-1, MMP-3, and MMP-9 proteins (all p > 0.05)ConclusionsIn TM cells, prostaglandin E2 agonist OMD did not increase the permeability through the TM cell monolayer, and the protein levels of MMPs. These suggest that the direct effect on the trabecular outflow by OMD may be limited.

Stanniocalcin-1 (STC-1), a downstream effector molecule in latanoprost signaling, acts independent of the FP receptor for intraocular pressure reduction.

Prostaglandin F2 alpha (PGF2α) analogues such as latanoprost are common first-line intraocular pressure (IOP) lowering medications. However, their clinical use is limited in some patient populations due to minimal or no IOP lowering response or side effects. In searching for a more targeted approach for IOP reduction, our lab recently identified Stanniocalcin-1 (STC-1) as a molecule that was required for latanoprost-mediated IOP reduction and also acted as a stand-alone IOP lowering agent. In order to determine whether latanoprost and STC-1 were equivalent and/or additive for IOP reduction, we treated C57BL/6J mice with one or a combination of these agents and measured IOP. Importance of the FP receptor for latanoprost- and STC-1-mediated IOP reduction was examined in C57BL/6J mice utilizing the pharmacologic FP receptor inhibitor AL-8810 as well as FP receptor knockout mice generated in our laboratory. Latanoprost-free acid (LFA) and STC-1 reduced IOP to a similar degree and were non-additive in C57BL/6J mice. As expected, the IOP lowering effects of LFA were abrogated by pharmacologic inhibition of the FP receptor with AL-8810 and in FP receptor knockout mice. In contrast, STC-1 maintained IOP-lowering effects in the presence of AL-8810 and also in FP receptor knockout mice. These results suggest that LFA and STC-1 show equivalent and non-additive IOP reduction in C57BL/6J mice and that unlike LFA, STC-1-mediated IOP reduction occurs independent of the FP receptor.

Correction: Ocular Hypotensive Effects of the ATP-Sensitive Potassium Channel Opener Cromakalim in Human and Murine Experimental Model Systems

Elevated intraocular pressure (IOP) is the most prevalent and only treatable risk factor for glaucoma, a leading cause of irreversible blindness worldwide. Unfortunately, all current therapeutics used to treat elevated IOP and glaucoma have significant and sometimes irreversible side effects necessitating the development of novel compounds. We evaluated the IOP lowering ability of the broad spectrum KATP channel opener cromakalim. Cultured human anterior segments when treated with 2 μM cromakalim showed a decrease in pressure (19.33 ± 2.78 mmHg at 0 hours to 13.22 ± 2.64 mmHg at 24 hours; p<0.001) when compared to vehicle treated controls (15.89 ± 5.33 mmHg at 0 h to 15.56 ± 4.88 mmHg at 24 hours; p = 0.89). In wild-type C57BL/6 mice, cromakalim reduced IOP by 18.75 ± 2.22% compared to vehicle treated contralateral eyes (17.01 ± 0.32 mmHg at 0 hours to 13.82 ± 0.37 mmHg at 24 hours; n = 10, p = 0.002). Cromakalim demonstrated an additive effect when used in conjunction with latanoprost free acid, a common ocular hypotensive drug prescribed to patients with elevated IOP. To examine KATP channel subunit specificity, Kir6.2(-/-) mice were treated with cromakalim, but unlike wild-type animals, no change in IOP was noted. Histologic analysis of treated and control eyes in cultured human anterior segments and in mice showed similar cell numbers and extracellular matrix integrity within the trabecular meshwork, with no disruptions in the inner and outer walls of Schlemm’s canal. Together, these studies suggest that cromakalim is a potent ocular hypotensive agent that lowers IOP via activation of Kir6.2 containing KATP channels, its effect is additive when used in combination with the commonly used glaucoma drug latanoprost, and is not toxic to cells and tissues of the aqueous humor outflow pathway, making it a candidate for future therapeutic development.

Ocular Hypotensive Effects of the ATP-Sensitive Potassium Channel Opener Cromakalim in Human and Murine Experimental Model Systems.

Elevated intraocular pressure (IOP) is the most prevalent and only treatable risk factor for glaucoma, a leading cause of irreversible blindness worldwide. Unfortunately, all current therapeutics used to treat elevated IOP and glaucoma have significant and sometimes irreversible side effects necessitating the development of novel compounds. We evaluated the IOP lowering ability of the broad spectrum KATP channel opener cromakalim. Cultured human anterior segments when treated with 2 μM cromakalim showed a decrease in pressure (19.33 ± 2.78 mmHg at 0 hours to 13.22 ± 2.64 mmHg at 24 hours; p<0.001) when compared to vehicle treated controls (15.89 ± 5.33 mmHg at 0 h to 15.56 ± 4.88 mmHg at 24 hours; p = 0.89). In wild-type C57BL/6 mice, cromakalim reduced IOP by 18.75 ± 2.22% compared to vehicle treated contralateral eyes (17.01 ± 0.32 mmHg at 0 hours to 13.82 ± 0.37 mmHg at 24 hours; n = 10, p = 0.002). Cromakalim demonstrated an additive effect when used in conjunction with latanoprost free acid, a common ocular hypotensive drug prescribed to patients with elevated IOP. To examine KATP channel subunit specificity, Kir6.2(-/-) mice were treated with cromakalim, but unlike wild-type animals, no change in IOP was noted. Histologic analysis of treated and control eyes in cultured human anterior segments and in mice showed similar cell numbers and extracellular matrix integrity within the trabecular meshwork, with no disruptions in the inner and outer walls of Schlemm’s canal. Together, these studies suggest that cromakalim is a potent ocular hypotensive agent that lowers IOP via activation of Kir6.2 containing KATP channels, its effect is additive when used in combination with the commonly used glaucoma drug latanoprost, and is not toxic to cells and tissues of the aqueous humor outflow pathway, making it a candidate for future therapeutic development.

緑内障点眼薬の薬物相互作用の解明と点眼薬物療法の費用最小化分析の確立

Latanoprost, a prostaglandin F2alpha analog (PG), has been shown to be an effective ocular hypotensive agent for treating glaucoma. Carbonic anhydrase (CA) inhibitors are also used to reduce ocular hypertension by decreasing aqueous humor secretion, and are given in combination with PG.Timolol malate has been shown to be an effective ocular hypotensive agent when used alone or with CA inhibitor on glaucoma patients.However, the effects of latanoprost and timolol malate on CA have not been clarified. Therefore, we studied the effects of latanoprost free acid and timolol hemihydrate on human CA (hCA)-I and hCA-II using stopped flow method.Latanoprost free acid inhibited the hydration activity of hCA-I or hCA-II by a noncompetitive mechanism. Timolol hemihydrate activates the enzyme activities of hCA-I and hCA-II. In hCA-I and hCA-II, the enzyme kinetic results clearly showed that timolol hemihydrate increases the value of Vmax but does not influence the value of Km. The noncompetitive inhibition mechanism and the binding mode of latanoprost free acid indicate that the behavior of latanoprost free acid is similar to that of simple anions. Timolol hemihydrate has a heterocyclic moiety and secondary amino group, which are typical structures in efficient activators of carbonic anhydrase.Moreover, we compared the cost of lowering intraocular pressure (IOP) by 1 mmHg in the treatment of glaucoma. Latanoprost is less expensive for lowering IOP by 1 mmHg than other products. These data should be helpful in selecting topical ophthalmic solutions from the viewpoint of cost-minimization.

Comparison of Efficacy and Safety Evaluation of Latanoprost Formulations with and without Benzalkonium Chloride

Background: This study investigated the safety (cytotoxicity in vitro) and pharmacological effects (ocular hypotensive effects and aqueous humor concentrations in normotensive monkeys in vivo) of latanoprost formulations with benzalkonium chloride (latanoprost with BAK) and without BAK (NP). Methods: A bioequivalence study of latanoprost with BAK and NP was also conducted on human healthy volunteers. Cytotoxicity and the protective effect against H2O2 stress in vitro were evaluated using human corneal epithelial cells. The ocular hypotensive effects in normotensive monkeys were measured by pneumatonometer and the aqueous humor concentrations of latanoprost free acid were determined by liquid chromatography/mass spectrum (LC/MS) methods. The bioequivalence study of latanoprost with BAK and NP was carried out as a single eye drop, two-sequence, crossover randomized study. Results: Cytotoxicity tests in vitro revealed that NP was less toxic than latanoprost with BAK and significantly inhibited H2O2 induced cell damage while latanoprost with BAK did not. The hypotensive efficacy and the latanoprost free acid concentrations in aqueous humor of each formulation were not significantly different in monkeys. In the bioequivalence study, NP was bioequivalent to latanoprost with BAK. NP was safer than latanoprost with BAK with respect the results obtained in the in vitro cytotoxicity test. There was no difference observed between latanoprost with BAK and NP in the IOP lowering effect in monkeys and healthy volunteers. Conclusion: Taken together, these results indicate that NP is as effective as latanoprost with BAK, and is more likely to maintain ocular surface health than latanoprost with BAK.

Unoprostone Isopropyl and Metabolite M1 Activate BK Channels and Prevent ET-1–Induced [Ca2+]iIncreases in Human Trabecular Meshwork and Smooth Muscle

Effects of cis-unoprostone isopropyl, its primary metabolite M1, trans-unoprostone isopropyl, latanoprost free acid, and fluprostenol were studied on Ca(2+)-activated K(+) (BK) channels, plasma membrane potential, [cAMP](i), [cGMP](i), and steady state [Ca(2+)](i), and protection against endothelin-1 (ET-1)-induced steady state [Ca(2+)](i) increases in human cortical neuronal (HCN-1A), trabecular meshwork (HTMC), and pulmonary artery smooth muscle (PASMC) cells. Effects on recombinant human prostaglandin (PG) receptors were determined. BK channel currents were measured using whole-cell patch clamp; [cAMP](i), [cGMP](i) with ELISAs; [Ca(2+)](i) with indo-1; plasma membrane potential using diBAC(4)(3); and PG receptor effects with PG receptor-expressing cells and FLIPR fluo-4 Ca(2+) assays. Unoprostone isopropyl and M1 activated sustained iberiotoxin (IbTX)-sensitive, AL-8810 (FP receptor antagonist)-insensitive BK channel currents with EC(50)s of 0.51 ± 0.03 nM (n = 5) and 0.52 ± 0.03 nM (n = 6) in HTMCs; 0.61 ± 0.06 nM (n = 8) and 0.46 ± 0.04 nM (n = 5) for M1 in HCN-1A cells and PASMC, respectively. They caused AL-8810-insensitive, IbTX-sensitive membrane hyperpolarization at 10 nM; up to 100 nM had no effect on or decreased [cAMP](i), [cGMP](i), and [Ca(2+)](i); and prevented ET-1-induced [Ca(2+)](i) increases. In contrast, 10 nM latanoprost free acid and fluprostenol caused membrane depolarization; increased [cAMP](i), [cGMP](i), and [Ca(2+)](i); and did not prevent ET-1-induced [Ca(2+)](i) increases. Trans-unoprostone isopropyl had no effects. Unoprostone isopropyl (1.25 μM) had no effect on PG receptors, and neither did M1, except for activating the FP receptor with EC(50) = 557.9 ± 55.2 nM (n = 4). Prostones, unoprostone isopropyl and M1, are potent AL-8810-insensitive, stereospecific BK channel activators, without [cAMP](i), [cGMP](i), or [Ca(2+)](i) involvement, and prevent ET-1-induced steady state Ca(2+) increases in HTMCs.

A Comparative Study of a Preservative-Free Latanoprost Cationic Emulsion (Catioprost) and a BAK-Preserved Latanoprost Solution in Animal Models

Benzalkonium chloride (BAK), a common preservative in eye drops, can induce ocular surface toxicity that may decrease glaucoma therapy compliance. The ocular hypotensive effect, pharmacokinetic (PK) profiles, and local tolerance of a preservative-free latanoprost 0.005% cationic emulsion (Catioprost(®)), and a BAK-preserved latanoprost 0.005% solution (Xalatan(®)), were compared. The ocular hypotensive effect was evaluated in monkeys with elevated intraocular pressure (IOP) induced by laser photocoagulation of the trabecular meshwork. Each monkey (n=8) received both latanoprost formulations once daily for 5 consecutive treatment days in a crossover design with at least a 2-week washout period between treatments. IOP was measured at baseline (on day 1, no instillation), on vehicle treatment day (day 0), and on treatment days 1, 3, and 5 before drug instillation and then hourly for 6 h. In rabbits, the ocular and systemic concentrations of latanoprost free acid were determined following a single instillation and the local tolerance of twice daily instillations over 28 days was assessed. Both the preservative-free and BAK-preserved latanoprost formulations shared the same efficacy profile with the maximum IOP reduction occurring 2 h after each morning dose (-15%, -20%, and -26%; -15%, -23%, and -23% on days 1, 3, and 5, respectively) and lasting through 24 h. The equivalence in efficacy was confirmed by the PK data demonstrating similar area under the curves (AUCs). While both formulations were well tolerated, the incidence of conjunctival hyperemia was reduced by 42% with the BAK-free latanoprost cationic emulsion. In animal models, a preservative-free latanoprost cationic emulsion was as effective as Xalatan(®) for lowering IOP with an improved ocular tolerance profile.

Development and validation of a liquid chromatography/electrospray ionization tandem mass spectrometry method for the quantification of latanoprost free acid in rabbit aqueous humor and ciliary body

A rapid, selective and sensitive method for quantification of latanoprost free acid in rabbit aqueous humor (AH) and ciliary body (CB) using reverse phase-high performance liquid chromatography coupled with electrospray ionization (ESI)-mass spectrometry/mass spectrometry has been developed and validated. Quantification in AH and CB was achieved by stable isotope dilution employing tetra-deuterated analog of latanoprost free acid, used as internal standard. Sample preparation was based on protein precipitation with methanol in AH, and on liquid extraction with a mixture of ethyl acetate and isopropanol 60:40 (v/v) in CB. Elution was achieved on an octylsilica (C8) column, using an isocratic elution method. Detection was performed on a triple quadrupole mass spectrometer, using ESI in positive ion selected reaction monitoring mode. Calibration curves were linear in the validated concentration ranges of 10-160 ng/mL in AH and 80-1280 ng/g in CB. The accuracy and precision values, obtained from three different sets of quality control samples, each analyzed in triplicate on three different days, were within the generally accepted criteria for analytical methods (< 15%). The limit of detection was 30.66 pg/mL in AH and 237.75 pg/g in CB. The assay proved to be accurate and precise when applied to the in vivo study of latanoprost free acid in rabbit AH and CB after single administration of an eye drops containing latanoprost.

Expression Profile of the Matricellular Protein Osteopontin in Primary Open-Angle Glaucoma and the Normal Human Eye

PURPOSE. To characterize the role of osteopontin (OPN) in primary open-angle glaucoma (POAG) and normal eyes. METHODS. OPN quantification was performed by enzyme-linked immunosorbent assay in aqueous humor (AH) obtained from human donor eyes (POAG and normal) and surgical samples (POAG and elective cataract removal). OPN expression and localization in whole eye tissue sections and primary normal human trabecular meshwork (NTM) cells were studied by Western blot and immunohistochemistry. Latanoprost-free acid (LFA)-treated NTM cells were analyzed for OPN gene and protein expression. Intraocular pressure was measured by tonometry, and central corneal thickness was measured by optical coherence tomography in young OPN(-/-) and wild-type mice. RESULTS. OPN levels were significantly reduced in donor POAG AH compared with normal AH (0.54 ± 0.18 ng/μg [n = 8] vs. 0.77 ± 0.23 ng/μg [n = 9]; P = 0.039). A similar trend was observed in surgical AH (1.05 ± 0.31 ng/μg [n = 20] vs. 1.43 ± 0.88 ng/μg [n = 20]; P = 0.083). OPN was present in the trabecular meshwork, corneal epithelium and endothelium, iris, ciliary body, retina, vitreous humor, and optic nerve. LFA increased OPN gene expression, but minimal change in OPN protein expression was observed. No difference in intraocular pressure (17.5 ± 2.0 mm Hg [n = 56] vs. 17.3 ± 1.9 mm Hg [n = 68]) but thinner central corneal thickness (91.7 ± 3.6 μm [n = 50] vs. 99.2 ± 5.5 μm [n = 70]) was noted between OPN(-/-) and wild-type mice. CONCLUSIONS. OPN is widely distributed in the human eye and was found in lower concentrations in POAG AH. Reduction of OPN in young mice does not affect IOP.

Expression and functional evidence of the prostaglandin F 2α receptor mediating contraction in human umbilical vein

Our purposes were to perform the pharmacological characterization of PGF 2α receptor (prostanoid FP-receptor) involved in human umbilical vein contraction and confirm its expression in this tissue. Umbilical cords from healthy patients after full-term deliveries were employed. The vein was dissected out of cords and used for either isolated organ bath or reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assays. The natural prostanoid FP-receptor agonist, PGF 2α, and its selective analogues, latanoprost and bimatoprost free acids are full agonists (produce more than 80% of the maximal contractile response to 5-HT) in human umbilical vein. The agonist potency (pEC 50) order was PGF 2α (6.01 ± 0.05) > latanoprost free acid (5.65 ± 0.07) = bimatoprost free acid (5.59 ± 0.08). The contractile effects of PGF 2α and latanoprost free acid were blocked competitively by the prostanoid FP-receptor antagonist, AL-8810. The antagonist potencies (p K B) of AL-8810 vs. PGF 2α (5.93 ± 0.05) and vs. latanoprost free acid (6.40 ± 0.08) in human umbilical vein are in good agreement with its ability to antagonize prostanoid FP receptors of rat, mouse and human cells. In all samples, clear signal was detected for cDNA amplification of prostanoid FP receptor and the specific prostanoid FP-receptor antibody recognized a protein of approximately 64 kDa. In conclusion, taking into account the obtained functional and biochemical data, we propose for the first time that human umbilical vein express prostanoid FP-receptors and these receptors could be involved in the vasoconstriction action of PGF 2α in this tissue.

The Mechanisms by Which Latanoprost Free Acid Inhibits Human Carbonic Anhydrase I and II

Latanoprost, a prostaglandin F2 alpha analogue, has been shown to be an effective ocular hypotensive agent when used alone on ocular hypertensive or open angle glaucoma patients. Carbonic anhydrase (CA) inhibitors are also used to reduce ocular hypertension by decreasing aqueous humor secretion, and are given in combination with prostaglandin F2 alpha analogue. It has been shown that prostaglandin F2 alpha, Minprostin F2 alpha, has been shown to increase the carbonic anhydrase (CA) activity and blood pressure. However, the effects of latanoprost on CA have not been clarified. Therefore, we studied the effects of latanoprost free acid on human carbonic anhydrase (HCA) I and II using the stopped flow method. Latanoprost free acid inhibited the hydration activity of HCA I or II by a noncompetitive mechanism. The inhibition constants (Ki) of latanoprost free acid for HCA I and II were 0.22 and 2.3 mM, respectively. Therefore, latanoprost free acid is a weak inhibitor of HCA I or II. AutoDock simulation of the latanoprost free acid-HCA I or II complex showed that the carboxylic moiety of latanoprost free acid, which is located at the end of the molecule, binds to the zinc ion of the active site by stretching of the chain of latanoprost free acid through the narrow and deep active site cavity of HCA I or II. In the active site cavity of HCA I or II, one side is hydrophilic and the other is hydrophobic. AutoDock simulation results clearly showed that latanoprost free acids lie down on the hydrophobic sides of the active site cavities in HCA I and II. The noncompetitive inhibition mechanism and the binding mode of latanoprost free acid indicate that the behavior of latanoprost free acid is very similar to that of simple anions.

Prostaglandins Increase Trabecular Meshwork Outflow Facility in Cultured Human Anterior Segments

To determine the effect of latanoprost free acid and prostaglandin E1 (PGE1) on outflow facility in cultured human anterior segments. Clinical studies find prostaglandin treatment increases uveoscleral outflow, but do not agree whether trabecular outflow increases. Cultured anterior segments eliminate the uveoscleral pathway and enable a direct assessment of trabecular outflow. Laboratory investigation. One anterior segment of an eye pair was placed in perfusion organ culture and received a continuous infusion of drug while the fellow anterior segment received vehicle. Histologic changes were assessed. Zymography and Western blots were used to analyze matrix metalloprotease (MMP) activity. Scleral hydraulic conductivities were measured. Latanoprost significantly increased outflow facility (67% +/- 11% vs control 6% +/- 10%, P < .001). Facility changes occurred within one hour of receiving drug, reaching a new baseline by 24 hours. Facility changes were reversible, requiring about 48 hours to return to pre-drug values. PGE1 caused less facility change (13% +/- 17% vs control 1% +/- 11%, P = .02). Prostaglandin treated anterior segments had regions of focal detachment and loss of Schlemm canal endothelial cells, with loss of extracellular matrix underlying some areas. MMPs were not consistently increased in Western blots, zymography, or immunohistochemistry. Scleral hydraulic conductivity increased, but not enough to account for total facility increase. Prostaglandins increase outflow facility in perfusion organ culture of human anterior segments. MMP activity was not consistently increased, and scleral hydraulic conductivity was not increased sufficiently to account for total facility increase. The histologic changes suggest a direct trabecular meshwork effect.

Bimatoprost: Mechanism of Ocular Surface Hyperemia Associated with Topical Therapy

Bimatoprost is a safe and well-tolerated intraocular pressure (IOP) lowering drug that was approved in the United States in 2001 for the treatment of glaucoma and ocular hypertension. It is highly efficacious and produces greater mean reductions in IOP than other currently available antiglaucoma drugs. Conjunctival hyperemia is a common side effect of bimatoprost, but the hyperemia is typically mild and transient. No association has been found between signs of inflammation and the presence of hyperemia in bimatoprost-treated patients. Preclinical studies have elucidated the pharmacological mechanism of bimatoprost-related hyperemia and have examined the possible involvement of inflammation. Bimatoprost, as well as the free acid of latanoprost, elicited endothelium-dependent vasorelaxation in the rabbit jugular vein preparation, a quantitative in vitro model for ocular surface hyperemia (OSH). The vasorelaxant responses to either bimatoprost or latanoprost free acid were significantly inhibited by L-NAME, a nitric oxide synthase inhibitor. Similarly, the in vivo OSH responses to topically applied bimatoprost or latanoprost in dog eyes were significantly inhibited by L-NAME. As predicted, prostaglandin E(2) (PGE(2))-induced conjunctival hyperemia was not inhibited by L-NAME, since PGE(2) has a direct relaxant effect on the vascular smooth muscle. In-life observations and histopathological assessment of ocular surface tissues following bimatoprost treatment were performed for multiple-dose one month, 6 month, or 12 month safety studies in rabbits, dogs, and non-human primates. Results of these studies showed no evidence of bimatoprost-related inflammation in the ocular surface tissues. In summary, OSH related to bimatoprost treatment in laboratory animals occurs by endothelial-derived nitric oxide-mediated vasodilatation and is not associated with inflammation. These studies suggest that conjunctival hyperemia, a side effect of bimatoprost treatment, results from non-inflammatory, pharmacologically based vasodilatation.

Vasoactive Responses of U46619, PGF2 , Latanoprost, and Travoprost in Isolated Porcine Ciliary Arteries

To compare the vasoactive properties of the prostanoids U46619 (thromboxane A2 analogue), prostaglandin F(2alpha) (PGF(2alpha)), latanoprost free acid, and travoprost free acid in isolated porcine ciliary arteries. In a myograph system (isometric force measurement), quiescent vessels were exposed (cumulatively) to U46619, PGF(2alpha), latanoprost, or travoprost (0.1 nM-0.1 mM). Experiments were also conducted in the presence of SQ 29548 (TP-receptor antagonist; 3-10 microM) or AL-8810 (FP-receptor antagonist; 3-30 microM). Contractions were expressed as the percentage of 100 mM potassium chloride-induced contractions. In quiescent vessels, contractions (concentration-response curves) induced by (0.1 mM) PGF(2alpha) (87.9% +/- 3.5%), U46619 (66.7% +/- 4.1%), and latanoprost (62.9% +/- 3.6%) were more pronounced (P < or = 0.001) than those induced by travoprost (23.0% +/- 4.4%). Concentration-response curves for PGF(2alpha), latanoprost, and travoprost were preceded by a smaller contraction peak (0.1 microM) that was higher (P < or = 0.05) for travoprost (24.4% +/- 2.8%) than for PGF(2alpha) (12.9% +/- 4.6%), but not different (P = 0.58) from latanoprost (22.0% +/- 3.0%). The 50% maximal contraction (PD50: negative log M concentration) of U46619 (-8.05 +/- 0.13) was lower (P < or = 0.001) than those of latanoprost (-5.65 +/- 0.10), PGF(2alpha) (-5.49 +/- 0.14), and travoprost (-5.12 +/- 0.52). Contractions were inhibited (P < or = 0.05-0.001) either by SQ 29548 or AL-8810. In isolated porcine ciliary arteries, all prostanoids tested induced contractions. Among them, travoprost appeared to be the least potent and U46619 the most efficient.