Julie Y. Crider

Ocular Hypotensive FP Prostaglandin (PG) Analogs: PG Receptor Subtype Binding Affinities and Selectivities, and Agonist Potencies at FP and Other PG Receptors in Cultured Cells

Natural prostaglandins (PGs) such as PGD2, PGE2, PGF2(2alpha), and PGI2 exhibited the highest affinity for their respective cognate receptors, but were the least selective agents when tested in receptor binding assays. Travoprost acid ([+]-fluprostenol) was the most FP-receptor-selective compound, exhibiting a high affinity (Ki = 35 +/- 5 nM) for the FP receptor, and minimal affinity for DP (Ki = 52,000 nM), EP1 (Ki = 9540 nM), EP3 (Ki = 3501 nM), EP4 (Ki = 41,000 nM), IP (Ki > 90,000 nM), and TP (Ki = 121,000 nM) receptors. Travoprost acid was the most potent PG analog tested in FP receptor functional phosphoinositide turnover assays in the following cell types: human ciliary muscle (EC50 = 1.4 nM), human trabecular meshwork (EC50 = 3.6 nM), and mouse fibroblasts and rat aortic smooth muscle cells (EC50 = 2.6 nM). Although latanoprost acid exhibited a relatively high affinity for the FP receptor (Ki = 98 nM), it had significant functional activity at FP (EC50 = 32-124 nM) and EP1 (EC50 = 119 nM) receptors. Bimatoprost acid was less selective, exhibiting a relatively high affinity for the FP (Ki = 83 nM), EP1 (Ki = 95 nM), and EP3 (Ki = 387 nM) receptors. Bimatoprost acid exhibited functional activity at the EP1 (EC50 = 2.7 nM) and FP (EC50 = 2.8-3.8 nM in most cells) receptors. Bimatoprost (nonhydrolyzed amide) also behaved as an FP agonist at the cloned human FP receptor (EC50 = 681 nM), in h-TM (EC50 = 3245 nM) and other cell types. Unoprostone and S-1033 bound with low affinity (Ki = 5.9 microM to > 22 microM) to the FP receptor, were not selective, but activated the FP receptor. In conclusion, travoprost acid has the highest affinity, the highest FP-receptor-selectivity, and the highest potency at the FP receptor as compared to the other ocular hypotensive PG analogs known so far, including free acids of latanoprost, bimatoprost, and unoprostone isopropyl ester.

Agonist activity of bimatoprost, travoprost, latanoprost, unoprostone isopropyl ester and other prostaglandin analogs at the cloned human ciliary body FP prostaglandin receptor.

We have determined the agonist activity of a number of natural prostaglandins and prostaglandin analogs at the FP prostaglandin receptor cloned from a human ciliary body cDNA library using phosphoinositide (PI) turnover assays. Travoprost acid (EC50 = 3.2 +/- 0.6 nM) was the most potent agonist in these cells followed by bimatoprost free acid (17-phenyl-trinor PGF2alpha; EC50 = 5.8 +/- 2.6 nM), fluprostenol (EC50 = 6.1 +/- 1.5 nM), and latanoprost free acid (PHXA85; EC50 = 54.6 +/- 12.4 nM) which was 17-fold weaker (p < 0.001) than travoprost acid. Unoprostone and S-1033 were significantly (p < 0.001) weaker than travoprost acid. The amide prodrug, bimatoprost (EC50 = 694 +/- 293 nM), activated this FP receptor with an intermediate potency. The isopropyl ester prodrugs, travoprost (EC50 = 42.3 +/- 6.7 nM), latanoprost (EC50 = 126 +/- 347 nM) and unoprostone isopropyl ester (EC50 = 9,100 +/- 2,870 nM), also exhibited FP agonist activity. However, other compounds such as PGI2, bradykinin, histamine, and serotonin were inactive. The agonist activities of bimatoprost, unoprostone (UF-021), fluprostenol and acids of travoprost and latanoprost were antagonized by AL-8810 (11beta-fluoro- 15-epi-15-indanyl-PGF2alpha), an FP-receptor-selective antagonist (Ki = 1.0 - 2.1 microM; n = 3). These studies have demonstrated, for the first time, agonist activities of the currently known and marketed ocular hypotensive prostaglandin analogs at the cloned human ciliary body FP prostaglandin receptor.

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.

Topical antibacterial therapy for mycobacterial keratitis: Potential for surgical prophylaxis and treatment

BACKGROUND Mycobacterium chelonae and Mycobacterium fortuitum are the 2 most commonly implicated species of nontuberculous mycobacteria in cases of bacterial keratitis. OBJECTIVES This article summarizes available data on the in vitro antibacterial activity against M chelonae or M fortuitum of 2 agents-amikacin and clarithromycin-that have been used in the treatment of bacterial keratitis. In addition, the article reviews the in vitro activity of 5 commercially available topical ocular fluoro-quinolones (in order of availability, ciprofloxacin, ofloxacin, levofloxacin, gatifloxacin, and moxifloxacin) that may have potential in the surgical prophylaxis and treatment of keratitis caused by M chelonae or M fortuitum. METHODS A search of the English-language literature indexed on the MEDLINE, Life Sciences, EMBASE, BIOSIS, and Pharmaprojects databases from 1966 to October 7, 2003, was conducted using the terms Mycobacterium chelonae, Mycobacterium fortuitum, bacterial keratitis, topical antibiotic therapy, ocular infection-mycobacteria, and LASIK infections. Data on the minimum concentrations at which 90% of isolates were inhibited (MIC(90)s) were reviewed and compared. RESULTS In the literature reviewed, the MIC(90) against M fortuitum was from 1 to 16 microg/mL for amikacin, from </=2 to >/=8 microg/mL for clarithromycin, from 0.1 to 1 microg/mL for ciprofloxacin, from 0.5 to 3.13 microg/mL for ofloxacin, and </=2 microg/mL for levofloxacin. The results were similar against M chelonae. The fourth-generation fluoroquinolones-gatifloxacin and moxifloxacin-had similar MIC(90)s against M fortuitum (both, 0.2 to 1 microg/mL); however, moxifloxacin had greater activity than gatifloxacin against M chelonae (minimum inhibitory concentration range: moxifloxacin, </=1 to 1.6 microg/mL; gatifloxacin, 3.2 to 6.25 microg/mL). CONCLUSIONS Topical fluoroquinolones may be beneficial for ocular surgical prophylaxis and for the treatment of keratitis caused by M chelonae or M fortuitum. Based on their reported MIC(90)s, none of the antibacterials reviewed had greater in vitro activity than moxifloxacin. In addition, moxifloxacin had greater in vitro activity than gatifloxacin against M chelonae, one of the predominant nontuberculous mycobacterial species involved in bacterial keratitis. Pending the conduct of controlled clinical studies, these findings suggest that moxifloxacin may have utility in the prevention and treatment of atypical mycobacterial keratitis.

Prostaglandin DP receptors positively coupled to adenylyl cyclase in embryonic bovine tracheal (EBTr) cells: pharmacological characterization using agonists and antagonists

Various prostaglandin agonists representing various classes of receptor subtypes were evaluated for their ability to stimulate adenylyl cyclase via the endogenous DP receptor in embryonic bovine tracheal (EBTr) cells. Two antagonists were used to block the agonist‐induced cyclic AMP production. ZK118182 (EC50=16±4 nM), RS‐93520 (EC50=23±4 nM), SQ27986 (EC50=33±9 nM), ZK110841 (EC50=33±5 nM), BW245C (EC50=59±19 nM) and PGD2 (EC50=101±10 nM) (n=4–70) were the most potent agonists. Whilst most compounds were full agonists (Emax=100% relative to PGD2), BW245C was significantly more efficacious than PGD2 (Emax=121±3%; P<0.001) and RS‐93520 appeared to be a partial agonist (Emax=64±9%; P<0.001). Agonists from the EP (e.g. enprostil; misoprostol; butaprost), FP (e.g. cloprostenol; fluprostenol; PHXA85), IP (iloprost; PGI2) and TP (U46619) prostanoid receptor classes were weak agonists or inactive in the EBTr cell assay system. The DP‐receptor antagonist, BWA868C, showed a competitive antagonist profile with pA2 values of 8.00±0.02 and 8.14±0.13 in Schild analyses with two structurally different agonists, BW245C and ZK118182, respectively (n=3). AH6809, another purported DP‐receptor antagonist, weakly inhibited PGD2‐ and ZK118182‐induced cyclic AMP production (Kis=808±193 nM and 782±178 nM, respectively). The current studies have characterized the DP receptor positively coupled to adenylyl cyclase in EBTr cells using a wide range of agonist and antagonist prostaglandins. These data support the utility of the EBTr cell line as a useful tool for the evaluation of DP receptor agonists and antagonists and for profiling other classes of prostaglandins.

Levobetaxolol (Betaxon™) and Other β-Adrenergic Antagonists: Preclinical Pharmacology, IOP-Lowering Activity and Sites of Action in Human Eyes

The pharmacological characteristics of levobetaxolol, a single active isomer of betaxolol, were determined and compared with activities of other beta-adrenoceptor antagonists. Levobetaxolol (43-fold beta1-selective) exhibited a higher affinity at cloned human beta1 (Ki = 0.76 nM) than at beta2 (Ki = 32.6 nM) receptors, while dextrobetaxolol was much weaker at both receptors. Levobetaxolol potently antagonized functional activities at cloned human beta1 and beta2 receptors, and also at guinea pig atrial beta1, tracheal beta2 and rat colonic beta3 receptors (IC50s = 33.2 nM, 2970 nM and 709 nM, respectively). Thus, levobetaxolol was 89-times beta1-selective (vs beta2). Levobetaxolol (Ki = 16.4 nM) was more potent than dextrobetaxolol (Ki = 2.97 microM) at inhibiting isoproterenol-induced cAMP production in human non-pigmented ciliary epithelial cells. Levobunolol and (l)-timolol had high affinities at beta1 and beta2 receptors but were considerably less beta1-selective than levobetaxolol. Levo-, dextro- and racemic-betaxolol exhibited little or no affinity, except at sigma sites and Ca2+-channels (IC50s > 1 microM), at 89 other receptor/ligand binding sites. Levobetaxolol exhibited a micromolar affinity for L-type Ca2+-channels. In conscious ocular hypertensive cynomolgus monkeys, levobetaxolol was more potent than dextrobetaxolol, reducing intraocular pressure by 25.9+/-3.2% at a dose of 150 microg/eye (n = 15-30). Quantitative [3H]-levobetaxolol autoradiography revealed high levels of binding to human ciliary processes, iris, choroid/retina, and ciliary muscles. In conclusion, levobetaxolol is a potent, high affinity and beta1-selective IOP-lowering beta-adrenoceptor antagonist.

3-Oxa-15-cyclohexyl Prostaglandin DP Receptor Agonists as Topical Antiglaucoma Agents

A series of prostaglandin DP agonists containing a 3-oxa-15-cyclohexyl motif was synthesized and evaluated in several in vitro and in vivo biological assays. The reference compound ZK 118.182 (9beta-chloro-15-cyclohexyl-3-oxa-omega-pentanor PGF(2alpha)) is a potent full agonist at the prostaglandin DP receptor. Saturation of the 13,14 olefin affords AL-6556, which is less potent but is still a full agonist. Replacement of the 9-chlorine with a hydrogen atom or inversion of the carbon 15 stereochemistry also reduces affinity. In in vivo studies ZK 118.182 lowers intraocular pressure (IOP) upon topical application in the ocular hypertensive monkey. Ester, 1-alcohol, and selected amide prodrugs of the carboxylic acid enhance in vivo potency, presumably by increasing bioavailability. The clinical candidate AL-6598, the isopropyl ester prodrug of AL-6556, produces a maximum 53% drop in monkey IOP with a 1 microg dose (0.003% w/w) using a twice-daily dosing regime. Synthetically, AL-6598 was accessed from known intermediate 1 using a novel key sequence to install the cis allyl ether in the alpha chain, involving a selective Swern oxidative desilylation of a primary silyl ether in the presence of a secondary silyl ether. In this manner, 136 g of AL-6598 was synthesized under GMP conditions for evaluation in phase I clinical trials.

Use of a semi-automated, robotic radioimmunoassay to measure cAMP generated by activation of DP-, EP2-, and IP-prostaglandin receptors in human ocular and other cell types

The aim of these studies was to compare the effects of several prostaglandin agonists on adenylyl cyclase activity in embryonic bovine tracheal (EBTr) cells, transformed human nonpigmented ciliary epithelial (NPE) cells and National Cancer Bank (NCB-20) cells. These cell types have been shown to express DP, EP2 and IP prostaglandin (PG) receptors, respectively. Cyclic AMP (cAMP) generation was measured by manual and semi-automated radioimmunoassay (RIA) techniques. ZK118182 (EC50 = 10-27 nM), PGE2 (EC50 = 21-27 nM) and PGI2 (EC50 = 3.5-4 nM) had the highest potency at the DP, EP2 and IP receptors, respectively. A plot of potency (EC50) values generated with both techniques showed a high degree of correlation for all three receptors. These studies provide further characterization of prostanoid receptor functional responses in three cell types and demonstrate the advantages of a semi-automated RIA method for the analysis of the second messenger cAMP.

Adenylyl Cyclase Activity Mediated by β-Adrenoceptors in Immortalized Human Trabecular Meshwork and Non-Pigmented Ciliary Epithelial Cells

Non-pigmented ciliary epithelial (NPE) and trabecular meshwork (TM) cells are important in maintaining normal aqueous humor dynamics through the inflow and outflow routes, respectively. The current studies were undertaken to evaluate the ability of several beta-adrenergic receptor agonists to stimulate various antagonists to inhibit cAMP production in cultured immortalized human TM and NPE cells using an automated enzyme immunoassay. Isoproterenol was the most potent agonist in both the NPE and TM cells. The rank order of potency of agonists in NPE and TM cells, respectively, was: isoproterenol [EC50 = 37 and 66 nM] > epinephrine [EC50 = 112 and 526 nM] > albuterol [EC50 = 426 and 785 nM] > norepinephrine [EC50 = 3 and > 10 microM] > phenylephrine [EC50 > 10 microM for both] = dopamine [EC50 > 10 microM for both](n = 3-19). The isoproterenol-induced cAMP production was inhibited by various antagonists with the following rank order of potency in NPE and TM cells, respectively: propranolol [Ki = 0.2 and 0.3 nM] = ICI-118551 [Ki = 0.5 and 0.4 nM] > levobunolol [Ki = 1.1 and 2.1 nM] > levobetaxolol [Ki = 13 and 14 nM] = racemic betaxolol [Ki = 43 and 19 nM] > dextrobetaxolol [Ki = 2,705 and 1,980 nM] > atenolol [Ki > 4,000 nM for both] (n = 3-7). These detailed pharmacological studies using a variety of agonists and antagonists further supported the presence of beta2-adrenergic receptors in immortalized human NPE and TM cells.

Cloned human 5-HT1A receptor pharmacology determined using agonist binding and measurement of cAMP accumulation

Twenty agonists and nine antagonists were evaluated for their ability to compete for [3H]‐8‐hydroxy‐2‐(di‐n‐propylamino)tetralin ([3H]‐8‐OH‐DPAT) binding to the cloned human serotonin‐1A (ch‐5‐HT1A) receptor expressed in Chinese hamster ovary cells and for their ability to alter adenylyl cyclase activity in the same cells. The most potent full agonists of high affinity included N,N‐dipropyl‐5‐carboxamidotryptamine (pEC50 = 9.6 ± 0.1), MDL 73005EF (pEC50 = 9.3 ± 0.2), 5‐methyl‐urapidil (pEC50 = 9.2 ± 0.1), 5‐carboxamidotryptamine (pEC50 = 9.1 ± 0.2), R(+)‐8‐OH‐DPAT (pEC50 = 8.6 ± 0.1) and BMY‐7378 (pEC50 = 8.6 ± 0.1). WB‐4101 (pEC50 = 8.3 ± 0.2; IA = 79%), clozapine (pEC50 = 8.1 ± 0.3; IA = 29%), (buspirone (pEC50 = 7.6 ± 0.2; IA = 79%), quipazine (pEC50<5; IA = 45%) and R‐DOI (pEC50<5; IA = 31%) were weaker agonists with partial agonist properties. The most potent antagonists were WAY‐100,635 (pKi = 10.2 ± 0.1), methiothepin (pKi = 8.8 ± 0.2), spiperone (pKi = 8.7 ± 0.2) and NAN‐190 (pKi = 8.5 ± 0.2). The receptor affinities and functional potencies were well correlated (r = 0.88; P<0.0001). Our binding data correlated well with the pharmacology of endogenous 5‐HT1A receptors in the rabbit iris‐ciliary body (r = 0.91; P<0.001) and rat hippocampus (r = 0.93, P<0.0001). Our functional cAMP data correlated well with other cAMP accumulation data (r = 0.8, P<0.01 vs calf hippocampus) but less so with [35S]‐GTP‐yS binding to the ch‐5‐HT1A receptor as a functional activity read‐out (r = 0.58, P<0.05). The present study provides a detailed pharmacological characterization of the ch‐5‐HT1A receptor using binding and functional assays.