Discovery and Preclinical Development of Novel Intraocular Pressure-Lowering Rho Kinase Inhibitor: Corticosteroid Conjugates.

Purpose: A new class of ocular steroids designed to mitigate steroid-induced intraocular pressure (IOP) elevation while maintaining anti-inflammatory activity was developed. Herein is described the discovery and preclinical characterization of ROCK'Ster compound 1. Methods: Codrugs consisting of a Rho kinase inhibitor (ROCKi) and a corticosteroid were synthesized. Compounds were initially screened in vitro for ROCKi activity and anti-inflammatory activity against the proinflammatory interleukin 23 and bacterial lipopolysaccharide (LPS) pathways. Selected compounds were then screened for solubility, chemical stability, and ex vivo corneal metabolism. Lead compound 1 was evaluated for IOP lowering in the Dutch Belted rabbit and for anti-inflammatory efficacy in both a postcataract surgery model and an allergic eye disease (AED) mouse model. Results: Several ROCK'Sters were found to be potent inhibitors of ROCK (Kis < 50 nM), have high anti-inflammatory activity in vitro (IC50s < 50 nM), display sufficient stability in topical ophthalmic formulations, and have a moderate rate of corneal metabolism. Compound 1 (0.1% and 0.25%, quater in die [QID]-4 times a day) demonstrated IOP-lowering capability without inducing hyperemia in our rabbit model. When compared with the marketed steroids, Durezol® and Pred Forte®, compound 1 (0.1%, 0.25%) demonstrated noninferiority in clinical scoring in a rabbit model of inflammation after surgery. In addition, anti-inflammatory outcomes were observed with compound 1 (0.1%) relative to Lotemax® or vehicle control in an AED mouse model. Conclusion: ROCK'Ster compound 1 is a novel compound suitable for topical ocular dosing that possesses IOP-lowering capability along with similar anti-inflammatory activity compared with marketed steroids.

Metallic versus Semiconducting SWCNT Chemiresistors: A Case for Separated SWCNTs Wrapped by a Metallosupramolecular Polymer.

As-synthesized single-walled carbon nanotubes (SWCNTs) are a mixture of metallic and semiconducting tubes, and separation is essential to improve the performances of SWCNT-based electric devices. Our chemical sensor monitors the conductivity of an SWCNT network, wherein each tube is wrapped by an insulating metallosupramolecular polymer (MSP). Vapors of strong electrophiles such as diethyl chlorophosphate (DECP), a nerve agent simulant, can trigger the disassembly of MSPs, resulting in conductive SWCNT pathways. Herein, we report that separated SWCNTs have a large impact on the sensitivity and selectivity of chemical sensors. Semiconducting SWCNT (S-SWCNT) sensors are the most sensitive to DECP (up to 10000% increase in conductivity). By contrast, the responses of metallic SWCNT (M-SWCNT) sensors were smaller but less susceptible to interfering signals. For saturated water vapor, increasing and decreasing conductivities were observed for S- and M-SWCNT sensors, respectively. Mixtures of M- and S-SWCNTs revealed reduced responses to saturated water vapor as a result of canceling effects. Our results reveal that S- and M-SWCNTs compensate sensitivity and selectivity, and the combined use of separated SWCNTs, either in arrays or in single sensors, offers advantages in sensing systems.

Ocular Hypotensive Response in Nonhuman Primates of (8R)-1-[(2S)-2-Aminopropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol-8-ol a Selective 5-HT2 Receptor Agonist.

Recently, it has been reported that 5-HT2 receptor agonists effectively reduce intraocular pressure (IOP) in a nonhuman primate model of glaucoma. Although 1-[(2S)-2-aminopropyl]indazol-6-ol (AL-34662) was shown to have good efficacy in this nonhuman primate model of ocular hypertension as well as a desirable physicochemical and permeability profile, subsequently identified cardiovascular side effects in multiple species precluded further clinical evaluation of this compound. Herein, we report selected structural modifications that resulted in the identification of (8R)-1-[(2S)-2-aminopropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol-8-ol (13), which displayed an acceptable profile to support advancement for further preclinical evaluation as a candidate for proof-of-concept studies in humans.

A single nanoscale junction with programmable multilevel memory.

Nanoscale devices that are sensitive to measurement history enable memory applications, and memristors are currently under intense investigation for robustness and functionality. Here we describe the fabrication and performance of a memristor-like device that comprises a single TiO2 nanowire in contact with Au electrodes, demonstrating both high sensitivity to electrical stimuli and high levels of control. Through an electroforming process, a population of charged dopants is created at the interface between the wire and electrode that can be manipulated to demonstrate a range of device and memristor characteristics. In contrast to conventional two-terminal memristors, our device is essentially a diode that exhibits memristance in the forward bias direction. The device is easily reset to the off state by a single voltage pulse and can be incremented to provide a range of controllable conductance states in the forward direction. Electrochemical modification of the Schottky barrier at the electrodes is proposed as an underlying mechanism, and six-level memory operations are demonstrated on a single nanowire.

FR-190997, a nonpeptide bradykinin B2-receptor partial agonist, is a potent and efficacious intraocular pressure lowering agent in ocular hypertensive cynomolgus monkeys.

Preclinical Research FR-190997 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl) cinnaminoacetyl]-N-methylamino]benzyloxy]-2-methyl-4- (2-pyridylmethoxy) quinoline), a nonpeptide bradykinin (BK) B2-receptor-selective agonist, represents a novel class of ocular hypotensive agents. FR-190997 exhibited a high affinity for the human cloned B2-receptor (Ki = 9.8 nM) and a relatively high potency (EC50 = 155 nM) for mobilizing intracellular Ca(2+) ([Ca(2+)]i) in human ocular cells from nonpigmented ciliary epithelium; trabecular meshwork [h-TM]; ciliary muscle [h-CM] that are involved in regulating intraocular pressure (IOP). Unlike BK, FR-190997 behaved as a partial agonist (Emax = 38-80%) in these cells and its [Ca(2+)]i-mobilizing effects were blocked by the B2-receptor-selective antagonists (HOE-140, Ki = 0.8-7 nM; WIN-64338, Ki = 157-425 nM). FR-190997 stimulated the production of prostaglandins (PGs) in h-CM and h-TM cells (EC50 = 15-19 nM; Emax = 27-33%); an effect that was reduced by the cyclooxygenase-2 inhibitor bromfenac, and by HOE-140. FR-190997 also induced pro-matrix metalloproteinase (MMP)-1 and MMP-3 release from h-CM cells. FR-190997 significantly lowered IOP (37% [P < 0.001] with 30 µg, 24 h post-topical ocular dosing) in ocular hypertensive eyes of conscious Cynomolgus monkeys. This effect was reduced by bromfenac and completely blocked by a B2-antagonist. FR-190997 primarily stimulated uveoslceral outflow (UVSO) of aqueous humor (2.6 to 3.9-fold above baseline). In conclusion, FR-190997 is a B2-receptor selective partial agonist that activates phospholipase C, mobilizes [Ca(2+)]; induces PG and pro-MMP production, and that profoundly lowers IOP by promoting UVSO in ocular hypertensive Cynomolgus monkey eyes.

In vivo optimization of 2,3-diaminopyrazine Rho Kinase inhibitors for the treatment of glaucoma.

A series of 2,3,6-pyrazine Rho Kinase inhibitors were optimized for in vivo activity for topical ocular dosing. Modifications of the 2-(piperazin-1-yl)pyrazine derivatives produced compounds with improved solubility and physicochemical properties. Modifications of the 6-pyrazine substituent led to improvements in in vitro potency. Compound 9 had the best in vitro and in vivo potency of EC50=260 nM with a 30% reduction of IOP in a non-human primate model at a dose of 0.33%.

Human non-pigmented ciliary epithelium bradykinin B2-receptors: receptor localization, pharmacological characterization of intracellular Ca(2+) mobilization, and prostaglandin secretion.

PURPOSE: To characterize the bradykinin (BK) receptor system in human non-pigmented ciliary epithelium (NPCE) using immunohistochemistry and functional cell-based techniques. METHODS: B2-receptor protein expression was studied in sections of human donor eyes and in Cynomolgus monkey eyes using immunohistochemical methods. The pharmacological characteristics of intracellular Ca(2+) ([Ca(2+)]i) mobilization in response to BK and related peptides, and blockade by two antagonists, was studied in primary human (p-h-NPCE) and in immortalized human NPCE (imh-NPCE) cells. Prostaglandins (PGs) release induced by BK was also studied in both cell-types using ELISA assays. Limited studies on primary human ciliary muscle (h-CM) cells and human trabecular meshwork (h-TM) cells and Chinese hamster ovary cells expressing human cloned B2-receptors (CHO-B2) were performed to compare with responses in both the NPCE cell-types. RESULTS: B2-receptor immunoreactivity was observed on human and Cynomolgus monkey NPCE cells on eye sections from both species. BK and related analog peptides differentially activated signaling mechanisms in NPCE cells by mobilizing [Ca(2+)]i, and the BK-evoked responses were blocked by B2-receptor-selective antagonists, HOE-140 and (S)-WIN-64338. Relative agonist potencies (EC50, nM) in p-h-NPCE cells [and in imh-NPCE cells] were: BK=3.4 ± 0.4 [6.3 nM]; Hyp(3)-BK EC50=1.7 ± 0.2 [6.0 nM], Lys-BK EC50=7.0 ± 0.3 [19.8 nM]; Met-Lys-BK EC50=106 ± 57.8 [125 nM]; Des-Arg(9)-BK EC50=>10,000 [16 µM]. The antagonist potencies for attenuating BK-induced mobilization of [Ca(2+)]i in these cells were: HOE-140 (Ki=7.9 ± 1.8 nM, n=4) and (S)-WIN-64338 (Ki=451 ± 44 nM, n=4). These NPCE cell data correlated well with those obtained for h-CM and h-TM cells, and with B2-receptor binding (r=0.99, p<0.0001). However, BK failed to stimulate total PGs production in both NPCE cell-types even though 10% bovine serum increased PG release (by 4.9-fold above baseline), and even though BK stimulated PG release from h-CM, h-TM and in CHO-B2 cells. BK (1 µM) also failed to increase nitric oxide (NO) levels in NPCE cells even though sodium nitropruside increased NO production by 3-fold. CONCLUSIONS: Human and monkey NPCE express immunoreactive B2-receptor proteins. These proteins were functionally active, since BK and related peptides potently stimulated mobilization of [Ca(2+)]i in p-h-NPCE and imNPCE cells that was blocked by two B2-selective antagonists. Down-stream signaling from B2-receptor activation did not appear to involve PG synthesis/release (or NO production) in NPCE cell-types under the present conditions, even though h-CM, h-TM and CHO-B2 cells exhibited robust PG synthesis and release in response to BK.

Trabecular meshwork bradykinin receptors: mRNA levels, immunohistochemical visualization, signaling processes pharmacology, and linkage to IOP reduction.

PURPOSE: To localize mRNA and protein of bradykinin (BK) receptors, BK precursor polypeptide (kininogen) mRNA, and to study functional biochemical pharmacology of the signal transduction processes mediated by B2-receptors in isolated human trabecular meshwork (h-TM) cells. Intraocular pressure (IOP) lowering effects of 2 kinins were also investigated. METHODS: Previously documented procedures were utilized throughout these studies. RESULTS: Kinninogen mRNA was most abundant in TM, ciliary body (CB), and optic nerve head and appeared elevated in glaucomatous h-TM tissue. High levels of B2-receptor mRNA were found in the sclera, iris, TM, and CB. B2-receptor subtype protein was localized in cells of the monkey and h-TM, and the treatment of isolated h-TM cells with transforming growth factor-ß2 (5 ng/mL) caused significant (P<0.04) downregulation of B2-receptor mRNA. In isolated primary h-TM cells, BK (EC50=0.8±0.2 nM; n=19) and Met-Lys-BK (EC50=6.5±1.5 nM) mobilized intracellular Ca(2+) and induced the release of prostaglandins (PGs) that was blocked by 2 B2-receptor antagonists [HOE-140; (S)-WIN-64338]. The cyclooxygenase inhibitor, bromfenac, abolished BK-induced PGs production. BK concentration dependently increased cell impedance, and it significantly (P<0.05) decreased h-TM cell volume in vitro. Intravitreal (ivt) administration of BK (50 µg), but not a B1-agonist (Sar-[D-Phe(9)]-Des-Arg(9)-BK; also at 50 µg), efficaciously lowered IOP (22.9% to 37% from baseline) of Dutch-Belted rabbits that naturally have high IOPs (27-28 mmHg). CONCLUSIONS: BK activates multiple signal transduction pathways in h-TM cells via B2-receptors that also mediate IOP reduction as observed in rabbits following ivt administration of BK. These ocular hypotensive effects of BK may be physiologically important and suggest a novel therapeutic potential of BK-related B2-agonists.

Protein expression, biochemical pharmacology of signal transduction, and relation to intraocular pressure modulation by bradykinin B2 receptors in ciliary muscle.

PURPOSE: To examine the bradykinin (BK) B2-receptor system in human and monkey ciliary muscle (CM) using immunohistochemical techniques, and to pharmacologically characterize the associated biochemical signal transduction systems in human CM (h-CM) cells. BK-induced modulation of intraocular pressure (IOP) in pigmented Dutch-Belt rabbits and cynomolgus monkeys was also studied. METHODS: Previously published procedures were used throughout these studies. RESULTS: The human and monkey ciliary bodies expressed high levels of B2-receptor protein immunoreactivity. Various kinins differentially stimulated [Ca²âº](i) mobilization in primary h-CM cells (BK EC50=2.4±0.2 nM > Hyp³,ß-(2-thienyl)-Ala5,Tyr(Me)8-(®)-Arg9-BK (RMP-7) > Des-Arg9-BK EC50=4.2 µM [n=3-6]), and this was blocked by B2-selective antagonists, HOE-140 (IC50=1.4±0.1 nM) and WIN-63448 (IC50=174 nM). A phospholipase C inhibitor (U73122; 10-30 µM) and ethylene glycol tetraacetic acid (1-2 mM) abolished the BK-induced [Ca²âº](i) mobilization. Total prostaglandin (primarily PGE2) secretion stimulated by BK and other kinins in h-CM cells was attenuated by the cyclooxygenase inhibitors bromfenac and flurbiprofen, and by the B2-antagonists. BK and RMP-7 (100 nM) induced a twofold increase in extracellular signal-regulated kinase-1/2 phosphorylation, and BK (0.1-1 µM; at 24 h) caused a 1.4-3.1-fold increase in promatrix metalloproteinases-1-3 release. Topical ocular BK (100 µg) failed to alter IOP in cynomolgus monkeys. However, intravitreal injection of 50 µg of BK, but not Des-Arg9-BK, lowered IOP in rabbit eyes (22.9±7.3% and 37.0±5.6% at 5 h and 8 h post-injection; n=7-10). CONCLUSIONS: These studies have provided evidence of a functional endogenously expressed B2-receptor system in the CM that appears to be involved in modulating IOP.

Single crystal iron nanocube synthesis via the surface energy driven growth method.

Single crystal iron nanocubes are produced by simply heating a bilayer film. This surface energy driven growth (SEDG) method exploits the difference in surface energies of the components (γ(Fe) ~ 2.2 J m(-2) versus γ(Nd) ~ 0.7 J m(-2)) in the binary alloy Fe-Nd system to produce nanocubes of the higher energy Fe component. The dimensions of the cubes range from tens to hundreds of nanometers in size and can be controlled by changing the initial thickness of iron in the deposited Fe-Nd bilayer prior to annealing at 700 °C. The composition and structure of the nanocubes was confirmed by transmission electron microscopy analysis as single crystal bcc iron in the α-phase. The cubes were found to exist as core-shell structures with the α-phase encased by an intermetallic Fe-Nd phase, characteristic of the SEDG growth mechanism.

Manipulating connectivity and electrical conductivity in metallic nanowire networks.

Connectivity in metallic nanowire networks with resistive junctions is manipulated by applying an electric field to create materials with tunable electrical conductivity. In situ electron microscope and electrical measurements visualize the activation and evolution of connectivity within these networks. Modeling nanowire networks, having a distribution of junction breakdown voltages, reveals universal scaling behavior applicable to all network materials. We demonstrate how local connectivity within these networks can be programmed and discuss material and device applications.

Cabergoline: Pharmacology, ocular hypotensive studies in multiple species, and aqueous humor dynamic modulation in the Cynomolgus monkey eyes.

The aims of the current studies were to determine the in vitro and in vivo ocular and non-ocular pharmacological properties of cabergoline using well documented receptor binding, cell-based functional assays, and in vivo models. Cabergoline bound to native and/or human cloned serotonin-2A/B/C (5HT(2A/B/C)), 5HT(1A), 5HT(7), alpha(2B), and dopamine-2/3 (D(2/3)) receptor subtypes with nanomolar affinity. Cabergoline was an agonist at human recombinant 5HT(2), 5HT(1A) and D(2/3) receptors but an antagonist at 5HT(7) and alpha(2) receptors. In primary human ciliary muscle (h-CM) and trabecular meshwork (h-TM) cells, cabergoline stimulated phosphoinositide (PI) hydrolysis (EC(50)=19+/-7 nM in TM; 76 nM in h-CM) and intracellular Ca(2+) ([Ca(2+)](i)) mobilization (EC(50)=570+/-83 nM in h-TM; EC(50)=900+/-320 nM in h-CM). Cabergoline-induced [Ca(2+)](i) mobilization in h-TM and h-CM cells was potently antagonized by a 5HT(2A)-selective antagonist (M-100907, K(i)=0.29-0.53 nM). Cabergoline also stimulated [Ca(2+)](i) mobilization more potently via human cloned 5HT(2A) (EC(50)=63.4+/-10.3 nM) than via 5HT(2B) and 5HT(2C) receptors. In h-CM cells, cabergoline (1 microM) stimulated production of pro-matrix metalloproteinases-1 and -3 and synergized with forskolin to enhance cAMP production. Cabergoline (1 microM) perfused through anterior segments of porcine eyes caused a significant (27%) increase in outflow facility. Topically administered cabergoline (300-500 microg) in Dutch-belted rabbit eyes yielded 4.5 microMM and 1.97 microM levels in the aqueous humor 30 min and 90 min post-dose but failed to modulate intraocular pressure (IOP). However, cabergoline was an efficacious IOP-lowering agent in normotensive Brown Norway rats (25% IOP decrease with 6 microg at 4h post-dose) and in conscious ocular hypertensive cynomolgus monkeys (peak reduction of 30.6+/-3.6% with 50 microg at 3h post-dose; 30.4+/-4.5% with 500 microg at 7h post-dose). In ketamine-sedated monkeys, IOP was significantly lowered at 2.5h after the second topical ocular dose (300 microg) of cabergoline by 23% (p<0.02) and 35% (p<0.004) in normotensive and ocular hypertensive eyes, respectively. In normotensive eyes, cabergoline increased uveoscleral outflow (0.69+/-0.7 microL/min-1.61+/-0.97 microL/min, n=13; p<0.01). However, only seven of the eleven ocular hypertensive monkeys showed significantly increased uveoscleral outflow. These data indicate that cabergoline's most prominent agonist activity involves activation of 5HT(2), 5HT(1A), and D(2/3) receptors. Since 5HT(1A) agonists, 5HT(7) antagonists, and alpha(2) antagonists do not lower IOP in conscious ocular hypertensive monkeys, the 5HT(2) and dopaminergic agonist activities of cabergoline probably mediated the IOP reduction observed with this compound in this species.

Pharmacological properties and discriminative stimulus effects of a novel and selective 5-HT2 receptor agonist AL-38022A [(S)-2-(8,9-dihydro-7H-pyrano[2,3-g]indazol-1-yl)-1-methylethylamine].

AL-38022A is a novel synthetic serotonergic (5-HT) ligand that exhibited high affinity for each of the 5-HT2 receptor subtypes (Ki100-fold less) affinity for other 5-HT receptors. In addition, AL-38022A displayed a very low affinity for a broad array of other receptors, neurotransmitter transport sites, ion channels, and second messenger elements, making it a relatively selective agent. AL-38022A potently stimulated functional responses via native and cloned rat (EC50 range: 1.9-22.5 nM) and human (EC50 range: 0.5-2.2 nM) 5-HT2 receptor subtypes including [Ca2+]i mobilization and tissue contractions with apparently similar potencies and intrinsic activities and was a full agonist at all 5-HT2 receptor subtypes. The CNS activity of AL-38022A was assessed by evaluating its discriminative stimulus effects in both a rat and a monkey drug discrimination paradigm using DOM as the training drug. AL-38022A fully generalized to the DOM stimulus in each of these studies; in monkeys MDL 100907 antagonized both DOM and AL-38022A. The pharmacological profile of AL-38022A suggests that it could be a useful tool in defining 5-HT2 receptor signaling and receptor characterization where 5-HT may function as a neurotransmitter.

Novel benzodifuran analogs as potent 5-HT2A receptor agonists with ocular hypotensive activity.

A series of 8-substituted benzodifuran analogs was prepared and evaluated for 5-HT(2A) receptor binding and activation. Several compounds containing ether and ester functionality were found to be potent agonists. Topical ocular administration of 5, 18, and 25 effectively reduced intra-ocular pressure in the hypertensive cynomolgus monkey eye in the range of 25-37%.

AL-34662: a potent, selective, and efficacious ocular hypotensive serotonin-2 receptor agonist.

PURPOSE AND METHODS: The aim of this study was to determine the ocular pharmacological characteristics of AL-34662 (1-((S)-2-aminopropyl)-1H-indazole-6-ol), a new synthetic serotonin-2 (5-HT2) receptor-agonist ocular hypotensive agent. A variety of well-documented in vitro and in vivo procedures were utilized to study the pharmacological attributes of AL-34662. RESULTS: AL-34662 exhibited a high affinity for the rat and human 5-HT2 receptor (IC50=0.8-1.5 nM) and for cloned human 5-HT2A-C receptors (IC50=3-14.5 nM). AL-34662 stimulated phosphoinositide turnover in human ciliary muscle (h-CM; EC50=289+/-80 nM) and in human trabecular meshwork (h-TM; EC50=254+/-50 nM) cells. AL-34662 also mobilized intracellular Ca2+ ([Ca2+]i) in h-CM (EC50=140+/-23 nM) and h-TM (EC50=38+/-8 nM) cells, being a full agonist like 5-HT itself. AL-34662's effects in the h-CM (and h-TM) cells were potently antagonized by 5-HT2A-antagonist M-100907 (IC50=1.8+/-0.7 nM), but weakly by 5-HT2B-antagonist (RS-127445 IC50>10 microM), 5-HT2B/C- antagonist (SB-242084 IC50=2.08 microM) and 5-HT2C antagonist (RS-102221 IC50>1 microM). AL-34662 caused relatively minimal ocular discomfort and hyperemia in rabbit and guinea pig eyes. It efficaciously lowered intraocular pressure (IOP) in the conscious ocular hypertensive monkey eyes (33% at 300 microg). The (R)-enantiomer (AL-34707) and the racemate (AL-34497) were less potent and/or efficacious than AL-34662 in all of these assays. CONCLUSIONS: AL-34662 is a high-affinity 5-HT2 receptor agonist that potently mobilizes [Ca2+]i in h-CM and h-TM cells, and which efficaciously lowers IOP in conscious ocular hypertensive cynomolgus monkey eyes through a local effect with minimal side-effects.

Preclinical pharmacology of AL-12182, a new ocular hypotensive 11-oxa prostaglandin analog.

PURPOSE: The aim of this study was to determine selected in vivo ocular properties of AL-12182 (5,6-dihydro-4,5-didehydro-11-deoxy-11-oxa-16-(3-chlorophenoxy)-omega-tetranor-PGF(2alpha) isopropyl ester) and the in vitro profile of its free acid, AL-12180. METHODS: Previously documented radioligand binding and functional assays involving human ciliary muscle cells (h-CM), human trabecular meshwork (h-TM) and other cells, and porcine ocular arteries were utilized. For in vivo procedures, we utilized rabbits, cats, and nonhuman primates to measure hyperemia, pupil diameter, and intraocular pressure (IOP), respectively. RESULTS: AL-12180 exhibited the highest affinity for the FP-receptor (K(i) = 143 +/- 36 nM) and much lower affinity for DP-, EP(3)-, IP-, and TP-receptors, and for several nonprostanoid receptors, enzymes, neurotransmitter uptake sites, ion channels, and other regulatory sites. AL-12180 activated phospholipase C-mediated phosphoinositide hydrolysis (potency, EC(50) = 13.7-42.7 nM) through the FP-receptor in a variety of cells, such as h-CM, h-TM cells, human embryonic kidney cells expressing the cloned human ciliary body FP-receptor (HEK-FP), mouse 3T3 cells, and rat vascular smooth muscle cells. AL-8810, an FP-antagonist, blocked the effects of AL-12180 in h-CM cells (IC(50) = 8.7 microM). AL-12180 also stimulated the mobilization of intracellular Ca(2+) ([Ca(2+)](i)) in h-TM cells (EC(50) = 111 +/- 36 nM), h-CM cells (EC(50) = 11 nM), and in host cells expressing the cloned human ciliary body FP-receptor (EC(50) = 5.9 +/- 3.1 nM). AL-12180 lacked significant agonist activity at DP-, EP(2)-, EP(4)-, IP-, and TP-receptors in cell-based assays. However, AL-12180 contracted porcine central retinal and short posterior ciliary arteries in vitro with micromolar potencies that appeared to involve TP-receptor activation. in vivo, AL-12182 elicited dose-related hyperemia in the rabbit eye, miosis in the cat eye, and ocular hypotension in the nonhuman primate eye. CONCLUSIONS: AL-12180 is a relatively potent and selective FP-receptor agonist whose isopropyl ester prodrug (AL-12182) lowers IOP by as much as 40% following topical ocular dosing in a laser-induced nonhuman primate model of ocular hypertension.

Human trabecular meshwork cells express functional serotonin-2A (5HT2A) receptors: role in IOP reduction.

PURPOSE: To apply a multidisciplinary approach to the identification and pharmacological characterization of the serotonin (5HT) receptors that mediate functional responses in human trabecular meshwork (h-TM) cells. To correlate in vitro findings with intraocular pressure (IOP) changes in conscious ocular hypertensive cynomolgus monkeys. METHODS: Documented methods were used, including reverse transcription-polymerase chain reaction (RT-PCR), phosphoinositide (PI) turnover, and intracellular Ca2+ ([Ca2+]i) mobilization. IOP was measured using standard applanation pneumatonometry. RESULTS: h-TM cells expressed robust mRNA signals for 5HT2A and 5HT2B receptors. 5HT and its analogues stimulated PI turnover and [Ca2+]i mobilization in h-TM cells from multiple donors (20/24 donors' TM cells responded). The agonist potencies (EC50) of compounds in mobilizing [Ca2+]i were (nM): 5-methoxy tryptamine, 8 +/- 4; (R)-DOI, 18 +/- 6; alpha-methyl-5HT, 22 +/- 3; 5HT, 40 +/- 7; 5-methoxy-dimethyl tryptamine, 64 +/- 27; and BW-723C86, 1213 +/- 210. These effects were potently blocked by the 5HT2A-receptor-selective antagonist, M-100907 (Ki = 1 +/- 0.3 nM), but weakly by antagonists of 5HT2B and 5HT2C receptors. Only 5HT2 receptor agonists such as (R)-DOI (300 microg lowered IOP 34.4% from baseline of 38.2 mm Hg; P < 0.001) and alpha-methyl-5HT (250 microg lowered IOP 30.8% from baseline of 41.8 mm Hg; P < 0.001) lowered IOP in ocular hypertensive cynomolgus monkeys. CONCLUSIONS: Strong signals for 5HT2A and 5HT2B receptor mRNAs were detected in h-TM cells. The receptors that coupled to PI hydrolysis and [Ca2+]i mobilization in h-TM cells were the 5HT2A receptor subtype, which also significantly lowered IOP in a primate model. These receptors may mediate the ocular hypotensive actions of 5HT2A agonists.

Pharmacological evidence for a functional serotonin-2B receptor in a human uterine smooth muscle cell line.

The present study investigated the serotonin-induced increase in phosphoinositide hydrolysis and mobilization of intracellular Ca2+ ([Ca2+]i) in human uterine smooth muscle cells (HUSMCs) to identify the serotonergic receptor positively coupled to phospholipase C in these cells. In phosphoinositide (PI) assays, serotonin (5-HT) and alpha-methyl-5-HT were potent, full agonists (EC50 = 20 and 4.1 nM, respectively), whereas the phenylethylamine, R-(-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride, was less active (EC50 = 63 nM). Proposed 5-HT2B-selective agonists, BW-723C86 [alpha-methyl-5-(2-thienylmethoxy)-1H-indole-3-ethanamine hydrochloride] and (+)-norfenfluramine, exhibited strong agonist potency and efficacy comparable with 5-HT (EC50 = 18 and 33 nM, respectively) and approximately 15-fold more potency than (-)-norfenfluramine (EC50 = 500 nM). 5-HT2C receptor agonists m-chlorophenylpiperazine and MK-212 [6-chloro-2-(1-piperaxinyl)pyrazine] were weak agonists in these cells, with potencies of 110 and 880 nM, respectively. A similar rank order of potency was observed in [Ca2+]i mobilization assays (r = 0.9, p < 0.005) in the HUSMC and with contraction of rat stomach fundus strips that contain a 5-HT2B receptor (r = 0.9, p < 0.001). Antagonist studies revealed that a 5-HT2B-selective antagonist, RS-127445 [2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyrimidine] (Ki = 0.13 nM), was significantly more effective at inhibiting 5-HT-induced activity than a 5-HT2A antagonist, M-100907 (R-(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidinemethanol]) (Ki= 914 nM) and the 5-HT2C antagonists RS-102221 (8-[5-(2,4-dimethoxy-5-(4-trifluoromethylsulfo-amido)phenyl-5-oxopentyl]-1,3,8-triazaspiro[4.5]decane-2,4-dione hydrochloride) (Ki = 2.5 microM) and SB-242084 (6-chloro-5-methyl-1-[6-92-methylpyridin-3-yloxy) pyridine-3-ylcarbamoyl] indoline) (Ki = 42.4 nM) in the HUSMC PI turnover assays. Taken together, these studies strongly suggest the presence of a functionally active 5-HT2B receptor subtype in HUSMCs. The physiological role of this receptor in these cells remains to be defined.

Serotonin-2 (5-HT2) receptor-mediated signal transduction in human ciliary muscle cells: role in ocular hypotension.

PURPOSE AND METHODS: The aim of this study was to characterize the serotonin (5-hydroxytryptamine; 5-HT) receptors that mediate phosphoinositide (PI) hydrolysis and intracellular Ca2+ ([Ca2+]i) mobilization in isolated cells of human ciliary muscle (h-CM) from multiple donors using a variety of agonists and antagonists. An additional aim was to visualize the mRNAs and receptor binding sites for 5-HT2 receptors in human ciliary body (h-CB), CM, and other tissues by reverse transcriptase polymerase chain reaction and quantitative autoradiography techniques, respectively, and to correlate with ocular hypotensive activity of such compounds. RESULTS: CBs isolated from several donor eyes revealed the presence of 5-HT(2A2C) receptor mRNAs. [3H]-5-HT and [3H]-ketanserin autoradiography on sections of human eyes revealed a high density of 5-HT2 receptor binding sites in the iris, ciliary epithelium, and longitudinal CM. In isolated h-CM cells, the agonists alpha-methyl-5-HT (EC50=63+/-17 nM), 5-HT (EC50=85+/-16 nM), (R)-DOI (EC50=165+/-47 nM), and 5-methoxy alpha-methyl tryptamine (EC50=1200+/-270 nM) differentially stimulated PI turnover. These agonists also mobilized [Ca2+]i in h-CM cells with the following potencies (EC50s): 5-methoxy-tryptamine=42+/-11 nM; alpha-methyl-5-HT=36+/-11 nM; (R)-DOI=120 nM; 5-HT=130+/-36 nM; MK-212=470 nM; mCPP>1 microM; BW723C86=1766 nM. The agonist-induced [Ca2+]i mobilization in h-CM cells was potently blocked by the 5-HT2A-selective antagonist M-100907 (IC50=1.2+/-0.4 nM) but less potently by the antagonists for 5-HT2B (RS-127445, IC50>10 microM) and 5-HT2C (RS-102221, IC50=5.8+/-2.3 microM) receptors. CONCLUSIONS: In conclusion, h-CB, h-CM, and CM cells express mRNAs and proteins for 5-HT2 receptor subtypes, of which the predominant functionally active subtype is the 5-HT2A receptor, as defined by agonist and antagonist activities. These receptors may be responsible for mediating the intraocular pressure reduction observed in recent literature with a number of 5-HT2 agonists, such as (R)-DOI, alpha-methyl-5HT, and AL-34662.

Human trabecular meshwork cell responses induced by bimatoprost, travoprost, unoprostone, and other FP prostaglandin receptor agonist analogues.

PURPOSE: To determine the functional agonist potencies of the intraocular pressure (IOP)-lowering prostaglandin F (FP)-class prostaglandin (PG) analogues (e.g., travoprost, latanoprost, bimatoprost, and unoprostone isopropyl ester) in human trabecular meshwork (h-TM) cells, by using phosphoinositide (PI) turnover and intracellular Ca(2+) ([Ca(2+)](i)) mobilization, and to confirm the FP nature of these receptors by using an FP receptor antagonist, 11beta-fluoro-15-epi-15-indanyl-PGF(2alpha) (AL-8810). METHODS: FP-receptor-mediated PI turnover and [Ca(2+)](i) mobilization were measured in h-TM cells by determining the accumulation of [(3)H]-inositol phosphates ([(3)H]-IPs) by anion-exchange chromatography and real-time fluorescence imaging, respectively. RESULTS: Various PG analogues concentration-dependently stimulated production of [(3)H]-IPs in h-TM cells with the following agonist potencies (median effective concentration; EC(50)): travoprost acid (EC(50) = 2.4 nM) > cloprostenol (EC(50) = 4.5 nM) > (+/-)-fluprostenol (EC(50) = 10.8 nM) > latanoprost acid (EC(50) = 34.7 nM) > bimatoprost acid (EC(50) = 112 nM) > PGF(2alpha) (EC(50) = 120 nM) >> unoprostone (UF-021; EC(50) = 3280 nM) > S-1033 (EC(50) = 4570 nM; all n = 3-9). Prodrug derivatives of these compounds exhibited the following potencies: travoprost (isopropyl ester; EC(50) = 89.1 nM) > latanoprost (isopropyl ester; EC(50) = 778 nM) > bimatoprost (amide; EC(50) = 1410-6940 nM). Travoprost acid, PGF(2alpha,) unoprostone, and S-1033 were tested in addition for [Ca(2+)](i) mobilization and found to have rapid and dose-dependent effects. The FP receptor-selective antagonist AL-8810 antagonized the (+/-)-fluprostenol-induced PI turnover in these cells (K(i) = 2.56 +/- 0.62 micro M) as well as that induced by bimatoprost and acids of latanoprost and travoprost. The agonist and antagonist potencies of the PG analogues from the PI turnover assays in h-TM cells correlated well with PI turnover data obtained from the cloned human ciliary body FP receptor (r = 0.92; P < 0.0001). CONCLUSIONS: The pharmacology of the h-TM cell FP-receptor-mediated PI turnover and [Ca(2+)](i) mobilization was defined using numerous synthetic (FP-selective) PG agonist analogues and an FP receptor antagonist, AL-8810. Bimatoprost, travoprost, latanoprost, unoprostone isopropyl ester, and their respective free acids were shown to be FP agonists in the h-TM cells.