Novel CCR3 Antagonists Are Effective Mono- and Combination Inhibitors of Choroidal Neovascular Growth and Vascular Permeability.

Choroidal neovascularization (CNV) is a defining feature of wet age-related macular degeneration. We examined the functional role of CCR3 in the development of CNV in mice and primates. CCR3 was associated with spontaneous CNV lesions in the newly described JR5558 mice, whereas CCR3 ligands localized to CNV-associated macrophages and the retinal pigment epithelium/choroid complex. Intravitreal injection of neutralizing antibodies against vascular endothelial growth factor receptor 2, CCR3, CC chemokine ligand 11/eotaxin-1, and CC chemokine ligand 24/eotaxin-2 all reduced CNV area and lesion number in these mice. Systemic administration of the CCR3 antagonists GW766994X and GW782415X reduced spontaneous CNV in JR5558 mice and laser-induced CNV in mouse and primate models in a dose-dependent fashion. Combination treatment with antivascular endothelial growth factor receptor 2 antibody and GW766994X yielded additive reductions in CNV area and hyperpermeability in mice. Interestingly, topical GW766994X and intravitreal anti-CCR3 antibody yielded strong systemic effects, reducing CNV in the untreated, contralateral eye. Contrarily, ocular administration of GW782415X in primates failed to substantially elevate plasma drug levels or to reduce the development of grade IV CNV lesions. These findings suggest that CCR3 signaling may be an attractive therapeutic target for CNV, utilizing a pathway that is at least partly distinct from that of vascular endothelial growth factor receptor. The findings also demonstrate that systemic exposure to CCR3 antagonists may be crucial for CNV-targeted activity.

Ocular hypotensive activity of BOL-303259-X, a nitric oxide donating prostaglandin F2α agonist, in preclinical models.

The aim of the study was to investigate the ocular hypotensive activity of a nitric oxide (NO)-donating latanoprost, BOL-303259-X, following topical administration. The effect of BOL-303259-X (also known as NCX 116 and PF-3187207) on intraocular pressure (IOP) was investigated in monkeys with laser-induced ocular hypertension, dogs with naturally-occurring glaucoma and rabbits with saline-induced ocular hypertension. Latanoprost was used as reference drug. NO, downstream effector cGMP, and latanoprost acid were determined in ocular tissues following BOL-303259-X administration as an index of prostaglandin and NO-mediated activities. In primates, a maximum decrease in IOP of 31% and 35% relative to baseline was achieved with BOL-303259-X at doses of 0.036% (9 µg) and 0.12% (36 µg), respectively. In comparison, latanoprost elicited a greater response than vehicle only at 0.1% (30 µg) with a peak effect of 26%. In glaucomatous dogs, IOP decreased from baseline by 44% and 10% following BOL-303259-X (0.036%) and vehicle, respectively. Latanoprost (0.030%) lowered IOP by 27% and vehicle by 9%. Intravitreal injection of hypertonic saline in rabbits increased IOP transiently. Latanoprost did not modulate this response, whereas BOL-303259-X (0.036%) significantly blunted the hypertensive phase. Following BOL-303259-X treatment, latanoprost acid was significantly elevated in rabbit and primate cornea, iris/ciliary body and aqueous humor as was cGMP in aqueous humor. BOL-303259-X lowered IOP more effectively than latanoprost presumably as a consequence of a contribution by NO in addition to its prostaglandin activity. The compound is now in clinical development for the treatment of glaucoma and ocular hypertension.

Antiglaucoma EP2 Agonists: A Long Road That Led Somewhere.

For >2 decades, EP2 agonists have been the subject of antiglaucoma research and development by scientists in industry and academia around the world. The road has led to the recent approval of the first drug of this class. This article reviews the development of EP2 agonists from conception to clinical approval, discussing pharmacology, structure, biodistribution, therapeutics, and drug delivery. An extensive list of source references is provided for the reader's benefit.

The prostanoid EP2 receptor agonist butaprost increases uveoscleral outflow in the cynomolgus monkey.

PURPOSE: To investigate the ocular hypotensive effect of the prostanoid EP2 receptor agonist butaprost and to establish its mechanism of action. METHODS: All experiments were performed in cynomolgus monkeys after topical application of butaprost (0.1%). The effects of butaprost on aqueous humor flow were determined by fluorophotometry. Total outflow facility was measured by the two-level, constant-pressure perfusion method, and uveoscleral outflow was determined by perfusion of FITC-labeled dextran through the anterior chamber. Effects on ocular morphology were studied after tissue fixation with transcardial perfusion by paraformaldehyde and immersion fixation of the globe, in animals subjected to long-term treatment with butaprost. Conscious ocular normotensive monkeys and monkeys with unilateral ocular hypertension were used for intraocular pressure (IOP) studies. RESULTS: Butaprost had no significant effect on aqueous humor flow or total outflow facility in ocular normotensive monkeys. Uveoscleral outflow was significantly higher in the butaprost treated eyes than in vehicle treated eyes, 1.03 +/- 0.20 vs. 0.53 +/- 0.18 microL.min(-1). After a 1-year treatment with butaprost, the morphology of the ciliary muscle was changed, showing increased spaces between ciliary muscle bundles and the apparent formation of new outflow channels. In many instances, changes were observed in the trabecular meshwork as well. Butaprost, in a single 0.1% dose, decreased IOP significantly in ocular normotensive monkeys and reduced IOP in laser-induced glaucomatous monkey eyes to the same level as that in the ocular normotensive contralateral eyes. CONCLUSIONS: The prostanoid EP2 receptor agonist butaprost appears to lower IOP by increasing uveoscleral outflow, according to both physiological and morphologic findings. Although the prostanoid EP2 receptor is structurally and functionally distinct from the FP receptor, the effects of EP2 and FP receptor stimulation on aqueous humor outflow are similar.

Intraocular Pressure-Lowering Activity of NCX 470, a Novel Nitric Oxide-Donating Bimatoprost in Preclinical Models.

PURPOSE: The prostaglandin F2alpha (PGF2α) analogue bimatoprost lowers intraocular pressure (IOP) by increasing uveoscleral outflow at doses shown to elicit redness of the eye. With the aim to enhance the IOP-lowering effect of bimatoprost we studied NCX 470 [(S,E)-1-((1R,2R,3S,5R)-2-((Z)-7-(ethylamino)-7-oxohept-2-enyl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl 6-(nitrooxy)hexanoate], a dual-acting compound combining bimatoprost with nitric oxide (NO) known to mainly act via relaxation of trabecular meshwork and Schlemm's canal. METHODS: New Zealand white rabbits with transient hypertonic saline-induced IOP elevation (tOHT-rabbits), cynomolgus monkeys with laser-induced ocular hypertension (OHT-monkeys), and normotensive dogs (ONT-dogs) were used. The levels of NCX 470, bimatoprost, and bimatoprost acid were determined in aqueous humor (AH), cornea (CR), and iris/ciliary body (ICB) by liquid chromatography-mass spectrometry/mass (LC-MS/MS), while cGMP in AH and ICB was monitored using an enzyme immunoassay (EIA) kit in pigmented Dutch Belted rabbits. RESULTS: NCX 470 (0.14%, 30 µL) lowered IOP in tOHT-rabbits with an E(max) of -7.2 ± 2.8 mm Hg at 90 minutes. Bimatoprost at equimolar dose (0.1%, 30 µL) was noneffective in this model. NCX 470 (0.042%, 30 µL) was more effective than equimolar (0.03%, 30 µL) bimatoprost in ONT-dogs (IOP change, -5.4 ± 0.7 and -3.4 ± 0.7 mm Hg, respectively, P < 0.05) and in OHT-monkeys (IOP change, -7.7 ± 1.4 and -4.8 ± 1.7 mm Hg, respectively, P < 0.05) at 18 hours post dosing. NCX 470 (0.042%, 30 µL) or bimatoprost (0.03%, 30 µL) resulted in similar bimatoprost acid exposure in AH, CR, and ICB while cGMP was significantly increased in AH and ICB at 18 and 24 hours after NCX 470 dosing. CONCLUSIONS: NCX 470 lowers IOP more than equimolar bimatoprost in three animal models of glaucoma by activating PGF2α and NO/cGMP signaling pathways.

Morphological changes in the anterior eye segment after long-term treatment with different receptor selective prostaglandin agonists and a prostamide.

PURPOSE: To investigate long-term changes in the anterior segment of primate eyes treated for one year with different prostaglandin agonists and a prostamide. The results were compared with those obtained after vehicle treatment and in untreated controls. METHODS: Sixteen young cynomolgus monkeys were unilaterally topically treated for 1 year with either bimatoprost 0.03% (prostamide), sulprostone 0.03% (EP(3)/EP(1) agonist), AH13205 0.1% (EP(2) agonist), or latanoprost 0.005% (FP agonist), which all lower IOP in this species at the doses applied. Four animals were treated with the vehicle only. In all cases the left eye was treated, the right eye remained untreated. Six monkeys served as untreated controls. Sections from 4 quadrants each of the circumference of the eyes of 16 drug-treated, 4 vehicle-treated and 6 untreated control animals were investigated qualitatively and quantitatively using light- and electronmicroscopy. The area of widened spaces between ciliary muscle bundles, the number of nerve fiber bundles at the muscle tips, and the width and length of the ciliary muscle were quantitated. RESULTS: The general morphology of the ciliary muscle and trabecular meshwork was normal in appearance and shape in all animals, whereas some localized morphologic changes were observed in the drug-treated animals. The changes were found to be similar in all four treatment groups. In the ciliary muscle, there was a significant increase in optically empty spaces between muscle bundles in the anterior portion of the longitudinal and the reticular ciliary muscle compared with untreated and vehicle-treated control animals. Within these spaces, significantly more myelinated nerve fiber bundles were found in drug-treated compared with normal control animals. Ultrastructurally the spaces were partly covered by endothelial-like cells which, in some areas, were in contact with the basement membrane of the microvasculature. In all treatment groups, there were also changes in the trabecular meshwork region. Significant regional differences among the different quadrants of the eyes and quantitative differences between treatment groups were observed. The ciliary epithelium had a normal appearance in all treatment groups. CONCLUSIONS: After one year of treatment with different prostaglandins and a prostamide, uveoscleral outflow pathways are enlarged and appear organized. Conventional outflow routes were also affected. Long-term treatment with AH13205, latanoprost, sulprostone, or bimatoprost also induces sprouting of nerve fibers.

Update on the mechanism of action of bimatoprost: a review and discussion of new evidence.

Bimatoprost is a pharmacologically unique and highly efficacious anti-glaucoma agent. It appears to mimic the activity of the prostamides, which are biosynthesized from the natural endocannabinoid anandamide by the enzyme cyclo-oxygenase 2 (COX-2). Bimatoprost has also been suggested to lower intraocular pressure by behaving as a prodrug or, alternatively, by stimulating FP receptors directly. These three distinctly different hypotheses for the mechanism of bimatoprost activity are discussed in the light of current evidence.

Bimatoprost effects on aqueous humor dynamics in monkeys.

The effects of bimatoprost on aqueous humor dynamics were quantified in monkey eyes. Uveoscleral outflow was measured by the anterior chamber perfusion method, using FITC-dextran. Total outflow facility was determined by the two-level constant pressure method. Aqueous flow was measured with a scanning ocular fluorophotometer. Uveoscleral outflow was 0.96 +/- 0.19 muL min(-1) in vehicle-treated eyes and 1.37 +/- 0.27 muL min(-1) (n = 6; P < .05) in eyes that received bimatoprost 0.01% b.i.d. x 5 days. Bimatoprost had no effect on total outflow facility, which was 0.42 +/- 0.05 muL min(-1) at baseline and 0.42 +/- 0.04 muL min(-1) after bimatoprost treatment. Bimatoprost had no significant effect on aqueous humor flow. This study demonstrates that bimatoprost increases uveoscleral outflow but not total outflow facility or aqueous humor flow, indicating that it lowers intraocular pressure in ocular normotensive monkeys by a mechanism that exclusively involves uveoscleral outflow.

A novel nitric oxide releasing prostaglandin analog, NCX 125, reduces intraocular pressure in rabbit, dog, and primate models of glaucoma.

PURPOSE: Nitric oxide (NO) is involved in a variety of physiological processes including ocular aqueous humor dynamics by targeting mechanisms that are complementary to those of prostaglandins. Here, we have characterized a newly synthesized compound, NCX 125, comprising latanoprost acid and NO-donating moieties. METHODS: NCX 125 was synthesized and tested in vitro for its ability to release functionally active NO and then compared with core latanoprost for its intraocular pressure (IOP)-lowering effects in rabbit, dog, and nonhuman primate models of glaucoma. RESULTS: NCX 125 elicited cGMP formation (EC(50) = 3.8 + or - 1.0 microM) in PC12 cells and exerted NO-dependent iNOS inhibition (IC(50) = 55 + or - 11 microM) in RAW 264.7 macrophages. NCX 125 lowered IOP to a greater extent compared with equimolar latanoprost in: (a) rabbit model of transient ocular hypertension (0.030% latanoprost, not effective; 0.039% NCX 125, Delta(max) = -10.6 + or - 2.3 mm Hg), (b) ocular hypertensive glaucomatous dogs (0.030% latanoprost, Delta(max)= -6.7 + or - 1.2 mm Hg; 0.039% NCX 125, Delta(max) = -9.1 + or - 3.1 mm Hg), and (c) laser-induced ocular hypertensive non-human primates (0.10% latanoprost, Delta(max) = -11.9 + or - 3.7 mm Hg, 0.13% NCX 125, Delta(max) = -16.7 + or - 2.2 mm Hg). In pharmacokinetic studies, NCX 125 and latanoprost resulted in similar latanoprost-free acid exposure in anterior segment ocular tissues. CONCLUSIONS: NCX 125, a compound targeting 2 different mechanisms, is endowed with potent ocular hypotensive effects. This may lead to potential new perspectives in the treatment of patients at risk of glaucoma.

Prostanoid EP4 receptor stimulation produces ocular hypotension by a mechanism that does not appear to involve uveoscleral outflow.

PURPOSE: As part of a systematic elucidation of the pharmacology of prostaglandin's (PG) effects on intraocular pressure in the monkey, the prototypical selective prostanoid EP(4) receptor agonist (3,7-dithia PGE(1)) was examined. It was found to be highly efficacious in nonhuman primates, and its mechanism of ocular hypotensive activity was investigated. METHODS: Intraocular pressure (IOP) was measured by pneumatonometry in conscious monkeys restrained in custom-designed chairs. All other animal experiments were performed in animals sedated with ketamine or anesthetized with ketamine/diazepam and given drug or vehicle for various lengths of time. Aqueous flow was determined by fluorophotometry. Total outflow facility was measured by the two-level, constant-pressure method and by 2-minute tonography in both normotensive and hypertensive monkey eyes. Uveoscleral outflow was measured by perfusing the anterior chamber with FITC-labeled dextran for 30 minutes at a fixed IOP of approximately 15 mm Hg. Isometric responses to drugs were measured in longitudinal and radial preparations of monkey and human isolated ciliary smooth muscle specimens. RESULTS: The selective EP(4) receptor agonist 3,7-dithia PGE(1) and an isopropyl ester prodrug thereof reduced IOP in monkeys. A single dose of 3,7-dithia PGE(1) isopropyl ester, at a 0.01% or 0.1% dose, decreased IOP in the glaucomatous monkey in the range of 40% to 50%. Studies on total outflow facility by the two-level, constant-pressure perfusion method and tonography indicated that EP(4) receptor stimulation facilitated aqueous humor outflow facility. No effect on aqueous flow was apparent. In contrast to all PGs and prostamides studied to date, 3,7-dithia PGE(1) exerted no effect on uveoscleral outflow measured directly. Moreover, it did not relax longitudinal or radial preparations of isolated human or monkey ciliary muscles. CONCLUSIONS: The EP(4) receptor agonist 3,7-dithia PGE(1) is a highly efficacious IOP-lowering drug in monkeys. It has no effect on uveoscleral outflow but does increase total outflow facility, which accounts for a substantial proportion of the ocular hypotensive activity.

Bimatoprost and prostaglandin F(2 alpha) selectively stimulate intracellular calcium signaling in different cat iris sphincter cells.

Bimatoprost is a synthetic analog of prostaglandin F(2 alpha) ethanolamide (prostamide F(2 alpha)), and shares a pharmacological profile consistent with that of the prostamides. Like prostaglandin F(2 alpha) carboxylic acid, bimatoprost potently lowers intraocular pressure in dogs, primates and humans. In order to distinguish its mechanism of action from prostaglandin F(2 alpha), fluorescence confocal microscopy was used to examine the effects of bimatoprost, prostaglandin F(2 alpha) and 17-phenyl prostaglandin F(2 alpha) on calcium signaling in resident cells of digested cat iris sphincter, a tissue which exhibits contractile responses to both agonists. Constant superfusion conditions obviated effective conversion of bimatoprost. Serial challenge with 100 nM bimatoprost and prostaglandin F(2 alpha) consistently evoked responses in different cells within the same tissue preparation, whereas prostaglandin F(2 alpha) and 17-phenyl prostaglandin F(2 alpha) elicited signaling responses in the same cells. Bimatoprost-sensitive cells were consistently re-stimulated with bimatoprost only, and prostaglandin F(2 alpha) sensitive cells could only be re-stimulated with prostaglandin F(2 alpha). The selective stimulation of different cells in the same cat iris sphincter preparation by bimatoprost and prostaglandin F(2 alpha), along with the complete absence of observed instances in which the same cells respond to both agonists, strongly suggests the involvement of distinct receptors for prostaglandin F(2 alpha) and bimatoprost. Further, prostaglandin F(2 alpha) but not bimatoprost potently stimulated calcium signaling in isolated human embryonic kidney cells stably transfected with the feline- and human-prostaglandin F(2 alpha) FP-receptor and in human dermal fibroblast cells, and only prostaglandin F(2 alpha) competed with radioligand binding in HEK-feFP cells. These studies provide further evidence for the existence of a bimatoprost-sensitive receptor that is distinct from any of the known prostaglandin receptor types.

Comparison of prostaglandin F2alpha, bimatoprost (prostamide), and butaprost (EP2 agonist) on Cyr61 and connective tissue growth factor gene expression.

Connective tissue growth factor (CTGF) and Cyr61 (cysteine-rich angiogenic protein 61) are members of the CCN gene family that encode multifunctional, extracellular matrix-associated signaling proteins. Because the mechanism of action of certain anti-glaucoma drugs involves extracellular matrix remodeling of ocular ciliary muscle, with a resultant increase in drainage of aqueous humor from the eye, we compared the effects of three pharmacologically distinct ocular hypotensive agents on Cyr61 and CTGF gene expression. Thus, prostaglandin F2alpha (PGF2alpha) (FP receptor agonist), Butaprost (EP2 receptor agonist), and Bimatoprost (a prostamide) were compared. Using Affymetrix gene chip technology, we first identified that PGF2alpha dramatically up-regulated Cyr61 and CTGF mRNA expression in HEK 293/EBNA cells (hFP-HEK 293/EBNA). Northern blot further confirmed the Cyr61 and CTGF up-regulation is in a dose- and time-dependent manner. PGF2alpha-induced up-regulation of Cyr61 appeared to exclusively involve the Rho pathway, and up-regulation of CTGF was via multiple intracellular pathways. Because prostamide receptors are, to date, defined only at the pharmacological level, Bimatoprost effects on Cyr61 and CTGF were studied in the isolated feline iris sphincter preparation, a tissue highly responsive to prostamides. Both PGF2alpha and Bimatoprost up-regulated Cyr61 mRNA expression in the cat iris tissue. Only PGF2alpha up-regulated CTGF mRNA expression in the cat iris. Therefore, PGF2alpha and Bimatoprost appear to interact with different receptors populations in the cat iris, according to their markedly different effects on CTGF. Activation of prostaglandin EP2 receptors (Gs-coupled) also up-regulated Cyr61 but not CTGF mRNA expression in the isolated cat iris. Similar data were observed in human primary ciliary smooth muscle cells. Thus, despite quite different signal transduction pathways, FP receptor stimulation up-regulates CTGF and Cyr61. The prostamide analog Bimatoprost and an EP2-selective agonist affects only Cyr61.