Serotonin-2B/2C Receptors Mediate Bovine Ciliary Muscle Contraction: Role in Intraocular Pressure Regulation.

PURPOSE: To study the pharmacological profile of the serotonin (5-hydroxytryptamine [5-HT]) receptor subtype mediating contractions in bovine isolated ciliary muscles. METHODS: Ciliary muscle strips were isolated from bovine eyeballs and mounted in organ baths containing aerated (95% O2, 5% CO2) Krebs buffer solution maintained at 37°C. Each muscle strip was attached at 1 end to a Grass Force-displacement Transducer connected to a Polyview Computer System for recording changes in isometric tension. After an equilibration period, ciliary muscle strips were exposed to selective agonists and antagonists of 5-HT receptors. RESULTS: Both selective and nonselective agonists for 5-HT produced concentration-dependent contractions of isolated ciliary muscles with the following rank order of potency: BW723C86>α-methyl-5-HT>MK-212>>8-hydroxy-DPAT>quipazine>R-DOI>>5-HT>>tryptamine. The selective 5-HT2 receptor antagonists, M-100907 (5-HT2A), RS-127445 (5-HT2B), and RS-102221 (5-HT2C), produced noncompetitive inhibition of the contractile effects of selective agonists yielding antagonist potency (pKB) values of 251 ± 27.2 nM (n = 4), 52.5 ± 6.3 nM (n = 4), and 79.4 ± 9.5 nM (n = 4), respectively. CONCLUSION: On the basis of the profile of activity of selective agonists and antagonists, we conclude that the 5-HT2B and 5-HT2C receptor subtypes appear to be the predominant serotonin receptors that mediate the contractile action of this amine in bovine isolated ciliary muscles.

Pharmacology of Serotonin Receptors Causing Contraction of Isolated Bovine Posterior Ciliary Arteries: Role in Ocular Blood Flow.

PURPOSE: To determine the serotonergic (5HT) receptor subtype mediating the contraction of bovine posterior ciliary arteries (BPCAs) in vitro. METHODS: Longitudinal isometric tension was measured in BPCA strips (4-5 mm) mounted in 25 mL organ baths containing oxygenated Krebs solution at 37°C. Cumulative contractile concentration-response (C-R) curves were generated for various 5-HT agonists to assess their potencies and maximal degrees of contraction. Multiple agonist C-R curves were also constructed in the presence and absence of receptor-selective antagonists to determine antagonist potencies using Schild plots. RESULTS: Selective and nonselective agonists for 5-HT receptors elicited concentration-dependent contractile responses in BPCAs with the following rank order of potency: MK-212 > BW723C86 > α-methyl-5-HT >5-methoxy-α-5-methyl-5-HT >> R-DO1 > >5-HT >> cabergoline >> 5-methoxy-dimethyl-tryptamine >> 2-methyl-5-HT >> tryptamine. Interestingly, both 8-OH-DPAT (5HT1A agonist) and quipazine (5HT3 agonist) did not elicit contractions in BPCAs. The contractions produced by BW723C86 (5-HT2B agonist) were antagonized by 5-HT receptor blockers, RS-127445 (5-HT2B antagonist), and M-100907 (5-HT2A antagonist), yielding antagonist pA2 values of 7.5 ± 0.12 (n = 4) and 6.2 ± 0.17 (n = 4), respectively. Furthermore, contractions elicited by MK-212 (5-HT2C agonist) was blocked by RS-102221 (5-HT2C antagonist), although noncompetitively. CONCLUSIONS: On the basis of the pharmacological profile of selective agonists and antagonists, we conclude that serotonin-induced contractions of the BPCA are mediated primarily by a combination of 5HT2C and/or 5HT2B receptors. It appears that 5-HT1A and 5-HT3 receptors are not involved in the contractile action of BPCAs to serotonin.

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%.

2,3-Diaminopyrazines as Rho kinase inhibitors.

Inhibition of rho kinase (ROCK) has been recognized as an important target for a number of diseases, including glaucoma. Herein we report SAR development around two hits from a kinase library that led to the discovery of the ROCK inhibitor compound 38. In vitro and in vivo analysis of this compound, including its effects in a monkey model of glaucoma will be discussed.

Molecular pharmacology of the DP/EP2 class prostaglandin AL-6598 and quantitative autoradiographic visualization of DP and EP2 receptor sites in human eyes.

DP-class prostaglandins and prostaglandin analogs (collectively, prostaglandins or PGs) such as PGD2, BW245C, ZK110841, and ZK118182, lower intraocular pressure (IOP) in animal models of ocular hypertension. A new analog of ZK118182 (AL-6556; 13,14-dihydro-ZK118182) was synthesized, and the isopropyl ester of AL-6556 (AL-6598) was shown recently to lower IOP in the ocular hypertensive cynomolgus monkey model of glaucoma and in human subjects. AL-6556 and AL-6598 had an affinity (Ki) of 2.66-4.43 microM for DP receptors but a much lower affinity (K(i)s = 38-103 microM) for EP3, FP, IP, and TP receptors (n = 3-5). In addition, AL-6556 and AL-6598 exhibited K(i)s > 100 microM for 19 nonprostanoid receptors. Both PGs stimulated cAMP production (EC50 = 1.07 +/- 0.1 microM and EC50 = 2.64 +/- 0.84 microM; n = 3) by way of DP receptors in embryonic bovine tracheal fibroblasts. While AL-6556 and AL-6598 were partial agonists (EC(50)s = 0.47-0.69 microM; E(max) = 35%-46%) at EP2 receptors in human nonpigmented epithelial cells, neither had any agonist activity at EP4, IP, or FP receptors. The DP antagonist, BWA868C, effectively antagonized the effects of AL-6556 with a high potency (IC50 = 22.8 +/- 3.9 nM; n = 3). DP receptors radiolabeled with [3H]BWA868C on human eye sections by quantitative autoradiography were highly concentrated in the ciliary process (CP), longitudinal (LCM) and circular (CCM) ciliary muscles, and iris with much lower specific binding in the cornea (CN), lens (LNS), and retina (RET). EP2 receptors labeled with [3H]PGE2 were concentrated in the LCM, CM, RET, and iris. In conclusion, AL-6598 and AL-6556 are relatively DP-receptor-selective PGs with full agonist activity at the DP and partial agonist activity at the EP2 receptor. The IOP-lowering activities of these compounds may involve both the inflow and outflow mechanisms, as DP and EP2 receptors were visualized in human ocular tissues involved in such aqueous humor dynamics.

Human ciliary muscle cell responses to FP-class prostaglandin analogs: phosphoinositide hydrolysis, intracellular Ca2+ mobilization and MAP kinase activation.

Phospholipase C induced phosphoinositide (PI) turnover, intracellular Ca(2+) ([Ca(2+)](i)) mobilization and mitogen-activated protein (MAP) kinase activation by FP-class prostaglandin analogs was studied in normal human ciliary muscle (h-CM) cells. Agonist potencies obtained in the PI turnover assays were: travoprost acid ((+)-fluprostenol; EC(50) = 2.6 +/- 0.8 nM) > bimatoprost acid (EC(50) = 3.6 +/- 1.2 nM) > (+/-)-fluprostenol (EC(50) = 4.3 +/- 1.3 nM) >> prostaglandin F(2 alpha) (PGF(2 alpha)) (EC(50) = 134 +/- 17 nM) > latanoprost acid (EC(50) = 198 +/- 83 nM) > S-1033 (EC(50) = 2930 +/- 1420 nM) > unoprostone (EC(50) = 5590 +/- 1490 nM) > bimatoprost (EC(50) = 9600 +/- 1100 nM). Agonist potencies in h-CM cells correlated well with those previously obtained for the cloned human ciliary body-derived FP receptor (r = 0.96, p< 0.001) and that present on h-TM cells (r = 0.94, p< 0.0001). Travoprost acid, PGF(2 alpha) and unoprostone also stimulated [Ca(2+)](i) mobilization in h-CM cells with travoprost acid being the most potent agonist. MAP kinase activity was stimulated in the h-CM cells with the following rank order of activity (at 100 nM): travoprost acid > PGF(2 alpha) > latanoprost acid > PGD(2) > bimatoprost > latanoprost = bimatoprost acid = fluprostenol > PGE(2) = S-1033 > unoprostone > PGI(2). The PI turnover, [Ca(2+)](i) mobilization and MAP kinase activation induced by several of these agonists was blocked by the FP receptor antagonist, AL-8810 (11 beta-fluoro-15-epiindanyl PGF(2 alpha)) (e.g. K(i) = 5.7 microM versus PI turnover). These studies have characterized the biochemical and pharmacological properties of the native FP prostaglandin receptor present on h-CM cells using three signal transduction mechanism assays and a broad panel of FP-class agonist analogs (including free acids of bimatoprost, travoprost and latanoprost) and the FP receptor antagonist, AL-8810.

Identification and characterization of the ocular hypotensive efficacy of travoprost, a potent and selective FP prostaglandin receptor agonist, and AL-6598, a DP prostaglandin receptor agonist.

The structure-activity studies that led to the identification of travoprost, a highly selective and potent FP prostaglandin analog, and AL-6598, a DP prostaglandin analog, are detailed. In both series, the 1-alcohol analogs are very effective and are thought to be acting as prodrugs for the biologically active carboxylic acids. The efficacy of amide prodrugs depends on the degree of substitution and the size of the substituents. Selected compounds are profiled in vitro and in vivo preclinically. Clinical studies show that travoprost 0.004% (isopropyl ester) provided intraocular pressure control superior to timolol 0.5% when used as monotherapy in patients with open-angle glaucoma or ocular hypertension. In clinical studies, AL-6598 0.01% provided a sustained intraocular pressure reduction with q.d. application; b.i.d. provided greater intraocular pressure control. The acute and, apparently, conjunctival hyperemia associated with topical ocular AL-6598 can be attenuated while maintaining intraocular pressure-lowering efficacy by formulating with brimonidine.