Human Cell Responses Induced by , , , and other FP Analogues

Najam A. Sharif, Curtis R. Kelly, and Julie Y. Crider

PURPOSE. To determine the functional agonist potencies of the sopropyl ester of prostaglandin F (FP)-class prosta- intraocular pressure (IOP)–lowering prostaglandin F (FP)–class Iglandin (PG) receptor , including travoprost,1 latano- prostaglandin (PG) analogues (e.g., travoprost, , bi- prost,2 and unoprostone isopropyl ester3 lower intraocular matoprost, and unoprostone isopropyl ester) in human trabec- pressure (IOP) in a number of mammalian species, including ular meshwork (h-TM) cells, by using phosphoinositide (PI) humans, and are used to treat and glauco- turnover and intracellular Ca2ϩ ([Ca2ϩ] ) mobilization, and to ma.4 Another , bimatoprost i 5 confirm the FP nature of these receptors by using an FP receptor (17-phenyl-trinor PGF2␣ ethyl amide), has also recently been antagonist, 11␤-fluoro-15-epi-15-indanyl-PGF ␣ (AL-8810). marketed for this indication. Even though putative FP recep- 2 6–8 2ϩ tors have been detected in the human ciliary muscle and METHODS. FP-receptor–mediated PI turnover and [Ca ] mobi- 9 i human trabecular meshwork (h-TM) cells, and an FP receptor lization were measured in h-TM cells by determining the accu- 10 3 3 from human ciliary body cDNA has been cloned, the detailed mulation of [ H]-inositol phosphates ([ H]-IPs) by anion-ex- pharmacologic characterization of the FP-receptor–mediated change chromatography and real-time fluorescence imaging, functional responses in these human ocular tissues and cells respectively. has not been described to date. In view of the paucity of this RESULTS. Various PG analogues concentration-dependently type of pharmacologic information, the purposes of our cur- stimulated production of [3H]-IPs in h-TM cells with the fol- rent studies were to determine the pharmacologic properties lowing agonist potencies (median effective concentration; of functionally coupled FP receptors in h-TM cells derived from ϭ Ͼ ϭ EC50): travoprost acid (EC50 2.4 nM) (EC50 several human donors without , by using selective FP Ͼ Ϯ ϭ Ͼ 4.5 nM) ( )-fluprostenol (EC50 10.8 nM) latanoprost receptor agonist prodrugs such as latanoprost and travoprost ϭ Ͼ ϭ Ͼ and their respective free acids; to assess the ability of some of acid (EC50 34.7 nM) bimatoprost acid (EC50 112 nM) ϭ ϾϾ ϭ these FP receptor agonists to mobilize intracellular Ca2ϩ PGF2␣ (EC50 120 nM) unoprostone (UF-021; EC50 3280 Ͼ ϭ ϭ ([Ca2ϩ] ) in h-TM cells; and to determine the antagonist effects nM) S-1033 (EC50 4570 nM; all n 3–9). Prodrug deriv- i atives of these compounds exhibited the following potencies: of a novel FP-receptor antagonist 11␤-fluoro-15-epi-15-indanyl- 11 ϭ Ͼ PGF ␣ (AL-8810) at the h-TM FP receptors, to complete the travoprost (isopropyl ester; EC50 89.1 nM) latanoprost 2 ϭ Ͼ characterization of these receptors. To our knowledge, this (isopropyl ester; EC50 778 nM) bimatoprost (amide; ϭ represents the first such detailed study of FP receptor pharma- EC50 1410–6940 nM). Travoprost acid, PGF2␣, unoprostone, 2ϩ cology in h-TM cells expressing endogenous FP prostaglandin and S-1033 were tested in addition for [Ca ]i mobilization and found to have rapid and dose-dependent effects. The FP recep- receptors. tor-selective antagonist AL-8810 antagonized the (Ϯ)-fluproste- ϭ Ϯ ␮ nol–induced PI turnover in these cells (Ki 2.56 0.62 M) MATERIALS AND METHODS as well as that induced by bimatoprost and acids of latanoprost and travoprost. The agonist and antagonist potencies of the PG The reagents were obtained from the cited sources as follows: all tissue analogues from the PI turnover assays in h-TM cells correlated culture media, antibiotics, supplements, and trypsin-EDTA from Life well with PI turnover data obtained from the cloned human Technologies (Grand Island, NY); fetal bovine serum from Hyclone ciliary body FP receptor (r ϭ 0.92; P Ͻ 0.0001). (Logan, UT); formic acid, ammonium formate, LiCl, and type B gelatin from Sigma Chemical Co. (St. Louis, MO); [3H]-myo-inositol from Am- CONCLUSIONS. The of the h-TM cell FP-receptor– 2ϩ ersham Corp. (Deerfield, IL); AG-1X8 anion-exchange resin from Bio- mediated PI turnover and [Ca ]i mobilization was defined Rad (Hercules, CA); scintillation fluid (Ecolume) from ICN Biomedicals using numerous synthetic (FP-selective) PG agonist analogues (Costa Mesa, CA); and bimatoprost, bimatoprost acid, (Ϯ)-fluprostenol and an FP receptor antagonist, AL-8810. Bimatoprost, tra- (1:1 mixture of [ϩ]) and [Ϫ] enantiomers), latanoprost, unoprostone, voprost, latanoprost, unoprostone isopropyl ester, and their and unoprostone isopropyl ester from Cayman Chemical Co. (Ann respective free acids were shown to be FP agonists in the h-TM Arbor, MI). Travoprost ([ϩ]-fluprostenol isopropyl ester), travoprost cells. (Invest Ophthalmol Vis Sci. 2003;44:715–721) DOI: free acid ([ϩ]), latanoprost acid, and AL-8810 were synthesized in the 10.1167/iovs.02-0323 Medicinal Chemistry Department of Alcon Research, Ltd. (Fort Worth, TX). Bimatoprost (Lumigan) was from Allergan, Inc. (Irvine, CA). S-1033 was generously provided by Shionogi (Osaka, Japan). FLIPR and the Ca2ϩ-sensitive dye kit were purchased from Molecular Devices From the Molecular Pharmacology Unit, Glaucoma Research, Al- con Research, Ltd., Fort Worth, Texas. Corp. (Menlo Park, CA). The h-TM cells were kindly provided by Mari Submitted for publication April 2, 2002; revised July 29, 2002; Engler, Therapeutic Target Research (Alcon Research, Ltd). accepted August 19, 2002. Commercial relationships policy: E, F. Cell Culture The publication costs of this article were defrayed in part by page 12 charge payment. This article must therefore be marked “advertise- The h-TM cells were obtained as previously described from dissected ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. TM explants of human donor eyes (from six different donors, ages 0.5, Corresponding author: Najam A. Sharif, Director and Head, Mo- 44, 51, 54, 80, and 85 years; all patients with no ocular disease history) lecular Pharmacology Unit, Alcon Research, Ltd. (R2-19), 6201 South kindly provided by various Eye Banks in the United States. The identity Freeway, Fort Worth, TX 76134-2099; [email protected]. of h-TM cells isolated from these explants was confirmed by a battery

Investigative Ophthalmology & Visual Science, February 2003, Vol. 44, No. 2 Copyright © Association for Research in Vision and Ophthalmology 715

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of biochemical and immunohistochemical techniques.12 The h-TM assays was defined as the compound concentration eliciting 50% of the cells were grown in DMEM with 1 g/L glucose supplemented with 100 maximal functional response induced by the agonist. Antagonist po- ␮ U/mL penicillin, 100 g/mL streptomycin, 2 mM L-glutamine, and 10% tency (equilibrium drug dissociation constants, Ki) was calculated with 11,17,18 ϭ ϩ fetal bovine serum. When confluent, these cells were subcultured and the following equation : antagonist Ki antagonist IC50/[1 11,17 seeded into uncoated 24-well plates for the phosphoinositide (PI)- (agonist concentration/agonist EC50)], where IC50 is the antagonist turnover experiments described. Cells were maintained in a humidified concentration causing 50% inhibition of the maximal functional re-

atmosphere of 5% CO2 and 95% air, with two changes of fresh medium sponse) when the antagonistic effects of multiple concentrations of weekly. Cells from passages 1 to 8 were used in the studies. AL-8810 were titrated against a fixed concentration of the different 2ϩ agonists used in the current studies. [Ca ]i mobilization fluorescence PI Turnover Assay traces obtained from the FLIPR-based assays were amalgamated by PI turnover assays of phospholipase C activity were conducted as using the graphics software, to show the concentration-dependent previously described and involved the measurement of agonist-stimu- nature of the agonist-stimulated responses. Ϯ lated production of [3H]-inositol phosphates ([3H]-IPs) by anion-ex- All data were calculated and represented as the mean SEM. A change chromatography.13,14 Briefly, confluent h-TM cells (ϳ50,000/ Student’s unpaired t-test was used to determine statistical differences well) were exposed for 24 to 30 hours to 3.8 ␮Ci [3H]-myo-inositol (if any) between the agonist potencies of the various compounds Ͻ (18.3 Ci/mmol) in 1.0 mL of the respective serum-free medium to label tested. P 0.05 was set as the minimum acceptable level of signifi- the cell membrane phospholipids. Cells were then rinsed once with cance between data sets. DMEM/Ham’s F-12 containing 10 mM LiCl before incubation with the agonist (or solvent as the control) in 1.0 mL of the same medium for 1 hour at 37°C, after which the medium was aspirated and 1 mL cold 0.1 RESULTS M formic acid was added. When the antagonist effects of AL-8810 were studied, it was added to the cells 15 minutes before exposure to the PI turnover in the h-TM cells was linear at least up to 90 agonist and the assay continued for another hour in the presence of the minutes (data not shown) and thus a 60-minute incubation antagonist. The chromatographic separation of [3H]-IPs on an AG-1X8 protocol was chosen for the agonist-induced generation of 3 resin-containing column was performed as previously described,13,14 [ H]-IPs, to maximize the signal-to-noise ratio and to reduce with sequential washes with water and 50 mM ammonium formate, possible hydrolysis or degradation of the test compounds. This followed by elution of the total [3H]-IPs fraction with 1.2 M ammonium assay paradigm was identical with that which has been used to formate containing 0.1 M formic acid. The eluate (4 mL) was collected characterize the FP-receptor–induced PI turnover in Swiss 3T3 13,14 and mixed with 15 mL scintillation fluid, and the total [3H]-IPs were mouse fibroblasts, rat vascular cells 11,16 determined by scintillation counting on a beta-counter at ϳ50% effi- (A7r5), and HEK-293 cells expressing the cloned human 15,19 ciency (LS6000; Beckman Instruments, Carlsbad, CA). ciliary body–derived FP receptor. In some typical experi- ments involving FP agonists such as (Ϯ)-fluprostenol, tra- Intracellular Ca2؉ Mobilization Assay voprost, and the free acids of latanoprost and travoprost, the amounts of [3H]-IPs generated in h-TM cells were as follows: FP receptor–mediated mobilization of intracellular Ca2ϩ ([Ca2ϩ] ) was i basal level, 1925 Ϯ 206 dissociations per minute (dpm; n ϭ 8); studied in h-TM cells with a fluorometric imaging plate reader (FLIPR; ␮ Ϯ 15,16 maximum stimulation (using 1–10 M agonists), 7780 1155 Molecular Devices Corp.), as previously described. In brief, h-TM dpm. Thus, there was a 4.0 Ϯ 0.36-fold stimulation of PI ␮ cells were transferred in a 50- L volume at a density of 50,000 cells per turnover above the basal level, thereby yielding a more than well to black-walled, 96-well tissue culture plates and cultured for two adequate signal-to-noise ratio. more days, to allow the cells to attach and flatten out in the culture The various prostaglandin analogues tested in the current plates. On the day of the experiment, one vial of FLIPR dye (Calcium studies stimulated accumulation of [3H]-IPs in h-TM cells in a Assay Kit; Molecular Devices Corp.) was resuspended in 50 mL of an concentration-dependent manner (Fig. 1) akin to that previ- FLIPR buffer consisting of Hanks’ balanced salt solution (HBSS), 20 mM ously reported for many other cell types expressing native or HEPES buffer, and 2.5 mM probenecid (pH 7.4). The h-TM cells were recombinant FP receptors.11,13–16 The relative potencies then loaded with the calcium-sensitive dye by addition of an equal (EC s) of these and other agonists are shown in Table 1. These ␮ 50 volume (50 L) to each well of the 96-well plate and incubated with data compared favorably with those reported for the FP recep- the dye for 1 hour at 23°C. The compound plate and cell plate were tor in several other cell types.11,13–16,19 The rank order of then placed in the FLIPR instrument. At the beginning of an experi- potency of the PG free acids tested in h-TM cells was: tra- mental run, a signal test was performed to check the basal fluorescence ϭ Ϯ Ͼ ϭ voprost acid (EC50 2.4 0.7 nM) cloprostenol (EC50 signal from the dye-loaded cells and the uniformity of the signal across Ϯ Ͼ Ϯ ϭ Ϯ Ͼ 4.5 1.3 nM) ( )-fluprostenol (EC50 10.8 2.1 nM) the plate. The basal fluorescence was adjusted to between 8,000 and ϭ Ϯ Ͼ latanoprost acid (EC50 34.7 2.4 nM) bimatoprost acid 12,000 units by modifying the exposure time, the camera F-stop, or the ϭ Ϯ Ͼ ϭ Ϯ ϾϾ (EC50 112 55 nM) PGF2␣ (EC50 120 26 nM) laser power. Instrument settings for a typical assay were the following: ϭ Ϯ Ͼ ϭ unoprostone (EC50 3280 1830 nM) S-1033 (EC50 laser power 0.3 to 0.6 W, camera F-stop F/2, and exposure time 0.4 4570 Ϯ 2280 nM). Many of the synthetic PGs (e.g., travoprost ␮ seconds. An aliquot (25 L) of the FP receptor agonist was then added acid, cloprostenol, (Ϯ)-fluprostenol) were more potent than ␮ to the existing 100 L dye-loaded cells at a dispensing speed of 50 the natural prostaglandin (PGF ). Similarly, the race- ␮ 2␣ L/sec. Fluorescence data were collected in real time at 1-second mate (Ϯ)-fluprostenol was nearly five times weaker than its intervals for the first 60 seconds and at 6-second intervals for an (ϩ)-enantiomer (travoprost acid; [ϩ]-fluprostenol; Table 1). As additional 120 seconds at 23°C. The functional responses were mea- expected, the prodrug derivatives of the former compounds sured and represented as peak fluorescence intensity minus basal exhibited lower potencies than their free acids, with the fol- intensity, in relative fluorescence units (RFUs) for each concentration lowing ranked order of potency observed: travoprost (isopro- of each agonist. ϭ Ϯ Ͼ pyl ester; EC50 89 20 nM) latanoprost (isopropyl ester; ϭ Ϯ Ͼ EC50 778 245 nM) 0.03% bimatoprost ophthalmic Data Analysis ϭ Ϯ Ն solution (Lumigan; Allergan Inc.; EC50 1410 397 nM) 2ϩ ϭ Ϯ The original PI turnover and [Ca ]i mobilization data were analyzed bimatoprost (amide; Cayman; EC50 6940 1836 nM; Fig. 1; with the nonlinear, iterative, sigmoidal curve-fitting function of a com- Table 1). The EC50s for bimatoprost from the two sources were mercial software program (Origin Scientific Graphics; Microcal Soft- statistically insignificant (P Ͻ 0.079). It was noteworthy that 13,14,17 Ϯ ware, Northampton, MA). Agonist potency (EC50) from these ( )-fluprostenol, latanoprost acid, bimatoprost acid, PGF2␣,

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unoprostone, and S-1033 exhibited a significantly (P Ͻ 0.02 to P Ͻ 0.001) lower potency than travoprost acid (Table 1). Similarly, travoprost (isopropyl ester) was significantly (P Ͻ 0.05 to P Ͻ 0.01) more potent than other prodrugs such as latanoprost and bimatoprost (Lumigan; Table 1). To study the downstream events after generation of IPs, limited experiments were conducted to study mobilization of 2ϩ [Ca ]i in the h-TM cells with a few of the FP receptor agonists of different potencies. As shown in Figure 2, travoprost acid, unoprostone, and S-1033 rapidly (within a few seconds) in- 2ϩ duced [Ca ]i mobilization in h-TM cells in a concentration- 2ϩ dependent manner. The [Ca ]i mobilization was a transient response measured only over a 3-minute period and thus rep- resented a nonequilibrium situation. However, once again, travoprost acid was found to be more potent than the other FP

agonists studied, based on their EC50 values: unoprostone, Ϯ Ϯ Ϯ 2400 656 nM; S-1033, 1080 220; PGF2␣, 98.6 26.7 nM; and travoprost acid, 26; 38 nM. Travoprost acid’s potency in 2ϩ the [Ca ]i mobilization assay compared well with that re- ϭ 15 ported for the mouse FP receptor (EC50 47 nM). To further characterize the pharmacology of the receptor responding to the aforementioned FP-class , we explored the antagonism of these responses by the FP-recep- 11 3 tor–selective antagonist, AL-8810. Here, AL-8810 concentra- FIGURE 1. Concentration-dependent stimulation of [ H]-IP formation Ϯ in h-TM cells induced by various FP receptor PG agonist analogues. The tion dependently antagonized the ( )-fluprostenol–induced PI 3 ϭ Ϯ ␮ h-TM cell membranes were radiolabeled with [ H]-myo-inositol after a turnover responses in the h-TM cells (Ki 2.56 0.62 M; 24- to 30-hour incubation. After cells were rinsed, different concentra- Fig. 3). In additional experiments, AL-8810 also antagonized PI ϭ ␮ tions of the agonists were added in culture medium containing 10 mM turnover induced by bimatoprost (Ki 1.0 M), travoprost LiCl for 1 hour at 37°C. The assays were terminated, and anion- ϭ ␮ ϭ ␮ acid (Ki 2.5 M), latanoprost acid (Ki 4.3 M), and exchange chromatography and liquid scintillation spectrometry were ϭ ␮ 3 unoprostone (Ki 2.4 M). The FP receptor antagonist po- used to isolate and quantify, respectively, the total [ H]-IPs. The data tency of AL-8810 at the cloned human ciliary body FP receptor were analyzed using a non-linear (sigmoidal-fit), iterative, curve-fitting ϭ ␮ was quite similar (Ki 1–2 M; described later). computer program to obtain the agonist potency values (EC50s) for each agonist (Table 1) and various concentration-response curves con- The agonist and antagonist potencies of these various pros- structed. The data are the mean Ϯ SEM of results in more than three taglandin analogues at the h-TM cell FP receptor, determined experiments, using normal h-TM cells isolated from TM tissues from up using the PI turnover assays, correlated well with their func- to six different human donors without ocular disease history. tional potencies determined at the cloned human ciliary body FP receptor (r ϭ 0.92; P Ͻ 0.0001; Fig. 4). In addition, the

TABLE 1. Agonist Potencies of FP-Class PG Analogues in h-TM Cells, Determined in PI-Turnover Assays

Functional Potency Statistical

Compound (EC50, nM) Significance

Travoprost acid ([ϩ]-fluprostenol) 2.4 Ϯ 0.7 Cloprostenol (acid) 4.5 Ϯ 1.3 NS* (Ϯ)-Fluprostenol (acid) 10.8 Ϯ 2.1 P Ͻ 0.02† Latanoprost acid (PHXA85) 34.7 Ϯ 2.4 P Ͻ 0.001† Ϯ Ͻ Bimatoprost acid (17-phenyl-trinor PGF2␣) 112 55 P 0.001† Travoprost (isopropyl ester) 89 Ϯ 20 Ϯ Ͻ PGF2␣ (acid) 120 26 P 0.001‡ Latanoprost (isopropyl ester) 778 Ϯ 245 P Ͻ 0.05§ Bimatoprost ophthlamic solution; amide (Lumigan) 1410 Ϯ 379 P Ͻ 0.05§ Unoprostone isopropyl ester 2310 Ϯ 1240 NS࿣ Unoprostone (acid; UF-021) 3280 Ϯ 1830 P Ͻ 0.001‡ Bimatoprost (amide) 6940 Ϯ 1830 P Ͻ 0.01§ S-1033 (acid) 4570 Ϯ 2280 P Ͻ 0.05‡

The h-TM cell membranes were radiolabeled with [3H]-myo-inositol after a 24- to 30-hour incubation. After the cells were rinsed, different concentrations of the agonists were added to them in culture medium containing 10 mM LiCl for 1 hour at 37°C. The assays were terminated, and anion exchange chromatog- raphy and liquid scintillation spectrometry were used to isolate and quantify the total [3H]-IPs, respec- tively. The data were analyzed using a non-linear (sigmoidal-fit), iterative, curve-fitting computer program Ϯ to obtain the agonist potency values (EC50s) for each agonist. Data shown are the mean SEM from three to nine independent PI-turnover experiments with h-TM cells obtained from up to six different human donors. * NS, not significantly different compared with travoprost acid potency. † Significantly different compared with travoprost acid potency. ‡ P Ͻ 0.001. § Significantly different compared with travoprost potency. ࿣ NS, not significantly different compared with travoprost potency.

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2ϩ FIGURE 2. [Ca ]i mobilization in h-TM cells in response to different FP receptor agonists. The h-TM cells were loaded with the Ca2ϩ-sensitive dye for 1 hour at 23°C and then ex- posed to different concentrations of the various FP receptor agonists on 2ϩ the FLIPR. Real-time [Ca ]i mobili- zation in the cells was monitored and recorded over 180 seconds at 23°C and the data analyzed on computer to obtain agonist potencies. Note the rapid concentration-dependent mo- bilization of Ca2ϩ in these cells by travoprost acid (A), unoprostone (B) and S-1033 (C). Similar data were ob- tained from other human donor TM cells and the cumulative agonist po- tencies calculated (see Results sec- tion).

FP-receptor–induced PI turnover data obtained from h-TM cells Specifically, whereas PGF2␣ was not a very potent com- also were highly correlated with data obtained from other cell pound in the h-TM PI turnover assay, various synthetic ana-

types and assays involving FP receptors as follows: h-TM versus logues of PGF2␣ were highly potent and efficacious agonists, mouse 3T3 cells, r ϭ 0.91; h-TM versus rat A7r5 cells, r ϭ 0.94; with travoprost acid, cloprostenol, and (Ϯ)-fluprostenol exhib- 3 ϭ h-TM versus [ H]-PGF2␣ binding, r 0.9 (data not shown). iting nanomolar potencies. The free acids of these PG ana- logues (e.g., travoprost acid, cloprostenol, (Ϯ)-fluprostenol, latanoprost acid, bimatoprost acid, and unoprostone) exhib- ISCUSSION D ited greater potencies than the respective isopropyl esters and The recent report of Anthony et al.9 provided initial evidence amide prodrug derivatives of these PGs, thus indicating that for the existence of putative FP receptors stimulating PI turn- the free acids represent the active moieties of these IOP- lowering agents. Travoprost acid ((Ϯ)-fluprostenol) was ap- over in h-TM cells, but only PGF2␣ was used to investigate the 9 proximately five times more active than the racemic (Ϯ)-flu- PI-turnover response. However, PGF2␣ binds to several differ- ent PG receptors and is not very selective1,18; hence, the prostenol in the PI turnover assays, and it was the most potent ϭ unequivocal identification of functionally active FP receptors agonist (travoprost EC50 2.4 nM) in the h-TM cells. This high in h-TM cells remained to be demonstrated. In the present functional potency of travoprost acid in the h-TM cells was also ϭ study, we extended these initial observations using h-TM cells observed at the cloned human ciliary body FP receptor (EC50 19 ϭ 1 obtained from eyes of a large number of donors and by using 3.2 nM) and the mouse FP receptor (EC50 2.7 nM). The ϭ several synthetic FP-class PG analogues (free acids, isopropyl potency of the natural prostaglandin agonist PGF2␣ (EC50 esters, and an amide), some of which have been shown to be 120 nM) at the h-TM FP receptor, detected by PI turnover, ϭ highly selective for the FP receptor and lower IOP in various matched that previously reported for h-TM cells (EC50 100 mammalian species, including humans. We also demonstrated nM),9 but unfortunately no other comparative data for other FP 2ϩ that many of the FP agonists also rapidly mobilized [Ca ]i in agonists in the h-TM cells are available in the literature. How- the h-TM cells. Furthermore, the FP-nature of the h-TM recep- ever, the relative agonist and antagonist potencies (and the tor responding to the PG analogues studied was confirmed by ranked order of potency) of the PG analogues studied in the the use of the FP-receptor antagonist, AL-8810.11,15,19 h-TM cells in our current studies were similar (r ϭ 0.92) to

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nique supported our current biochemical and pharmacologic functional studies on the h-TM cell FP receptor. Cultured h-TM 22 cells produce PGE2 and PGF2␣ and FP-receptor–mediated 23 generation of PGE2 has been reported. Because these endog- enous PGs have been shown to regulate aqueous humor dy- namics in anterior segments,4,24 functional activa- tion of the h-TM cell FP receptors may therefore play a central role in the PG-mediated regulation of IOP by promoting con- ventional outflow in addition to the uveoscleral outflow. In addition, because production of matrix metalloproteinases (MMPs) by ciliary muscle cells in response to FP agonists has been implicated as a mechanism of action for latanoprost,25 it is possible that the FP receptor in h-TM cells has a similar role. However, further work is needed to explore this possibility. The prodrug bimatoprost has recently become available to treat ocular hypertension.5 Unoprostone isopropyl ester3,26 is another PG analogue prodrug that has been available for the same indication. Recent reports have suggested that bimato- prost5 and unoprostone isopropyl ester27,28 do not interact with any PG receptors to induce biological responses. How- ever, the present studies in h-TM cells provide strong evidence for the FP receptor agonist nature of both of these compounds and their respective free acids in both the PI turnover assay and 2ϩ [Ca ]i mobilization assays. These data in h-TM cells have FIGURE 3. Concentration-dependent inhibition of (Ϯ)-fluprostenol– induced formation of [3H]-IPs formation by AL-8810 in normal h-TM cells isolated from numerous human donors. The h-TM cell membranes were radiolabeled with [3H]-myo-inositol after a 24-hour incubation. After rinsing the cells, different concentrations of the FP receptor antagonist, AL-8810, were added to the cells in the culture medium containing 10 mM LiCl and the incubation continued for 15 to 20 minutes at 37°C. After this time, the agonist, (Ϯ)-fluprostenol (100 nM final), was added to the cells and the incubation continued for another hour at 37°C. The assays were terminated and anion-exchange chro- matography and liquid scintillation spectrometry were used to isolate and quantify, respectively, the total [3H]-IPs. The data were analyzed using a non-linear (sigmoidal-fit), iterative, curve-fitting computer pro- gram to obtain the antagonist potency value for AL-8810 and the concentration-response curves constructed as shown. Data are the mean Ϯ SEM of results from more than three experiments. Similar data were obtained for the antagonism by AL-8810 of the PI turnover induced by bimatoprost, unoprostone, and the other FP-receptor PG agonist analogues described in the Results section.

those found for the FP receptor cloned from the human ciliary body.10,19 Furthermore, the relatively high degree of correla- tion between the potencies of up to eight FP agonists and an antagonist at the h-TM cells and those at the mouse13,14 and rat16 FP receptor supported the close homology of the amino acid sequences (and intracellular signaling mechanisms) of the FP receptor in these different species, determined by molecu- lar biological techniques.20 It was considered important to study the downstream sig- nal-transduction events related to FP receptor agonist–induced PI turnover. PI hydrolysis is known to result in release of Ca2ϩ from intracellular stores.9,13,15 Accordingly, we observed a 2ϩ rapid and concentration-dependent mobilization of [Ca ]i in h-TM cells by several FP agonists, including travoprost acid, IGURE 2ϩ F 4. Correlation of the agonist and antagonist potencies of nu- unoprostone, and S-1033. Even though the [Ca ]i mobiliza- merous prostaglandin analogues of the FP-class at the FP receptors in tion assay is conducted under transient and nonequilibrium normal h-TM cells and at the cloned human ciliary body derived FP conditions because of the real-time nature of this technique, receptor. Agonist potencies of various FP analogues (and antagonist with observations being made over seconds,15 the potencies of potencies of AL-8810) were determined in normal primary h-TM cells the compounds obtained from the PI turnover assay ([3H]-IP from several human donors and in HEK-293 cells expressing the cloned 15,19 accumulation over 60 minutes) and the latter [Ca2ϩ] assay human ciliary body FP receptor using the PI turnover assay. The i Ϫlog of the EC (pEC ) and –log of the K (pK ) for the 14 prosta- were of similar magnitude. More important, travoprost acid 50 50 i i 2ϩ glandins (13 agonists and 1 antagonist) were calculated to construct was still the most potent and efficacious agonist in the [Ca ]i the correlation plot. Note the high degree of correlation between the mobilization assay in the h-TM cells, akin to the PI-turnover two different FP receptor systems (r ϭ 0.92; P Ͻ 0.0001). High assay findings. correlations were also obtained between the h-TM cells and other cells The recent detection of FP receptor mRNA in human TM derived from mouse and rat tissues expressing endogenous FP recep- biopsy specimens21 and TM cells9 determined by RT-PCR tech- tors (see Results section).

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further substantiated and underscored other recent findings of epithelial and ciliary muscle cells. Biochem Pharmacol. 1997;53: FP agonist effects of bimatoprost and its free acid (and also of 1249–1255. unoprostone; UF-021) at the cloned human ciliary body FP 7. Sharif NA, Davis T, Williams GW. [3H]-AL-5848 ([3H] 9-␤-(ϩ) receptor.15,19,29 Bimatoprost free acid (17-phenyl-trinor fluprostenol): carboxylic acid of travoprost (AL-6221), a novel FP prostaglandin to study the pharmacology and autoradiographic PGF2␣) has also been shown to be an agonist at the constitutive mouse13,14 and rat16 FP receptor and the recombinant human localization of the FP receptor. J Pharmac Pharmacol. 1999;51: ocular FP receptor,19,29 in PI turnover assays. Similarly, uno- 685–694. 8. Davis TL, Sharif NA. Quantitative autoradiographic visualization prostone (UF-021; 13,14-dihydro-15-keto-20-ethyl-PGF2␣) com- 3 and pharmacology of FP-prostaglandin receptors in human eyes petes for [ H]-PGF2␣ binding to bovine corpus luteum FP receptors and also behaves as an FP receptor agonist at the using the novel phosphor-imaging technology. J Ocul Pharmacol mouse14 and human19 FP receptor, stimulating PI turnover and Ther. 1999;15:323–336. 2ϩ mobilizing intracellular Ca . Therefore, both bimatoprost and 9. Anthony TL, Pierce, KL, Stamer WD, Regan JW. Prostaglandin F2␣ unoprostone bind to and activate FP prostaglandin receptors to receptors in the human trabecular meshwork. Invest Ophthalmol induce second-messenger functional responses. Vis Sci. 1998;39:315–321. Travoprost acid ([ϩ]-fluprostenol) exhibited a greater over- 10. Kunapuli P, Lawson JA, Rokach J, FitzGerald GA. Functional char- acterization of the ocular prostaglandin F (PGF ) receptor. J Biol all efficacy than bimatoprost and unoprostone in the h-TM cell 2␣ 2␣ PI turnover assays (Fig. 1) with general confirmation in the Chem. 1997;272:27147–27154. h-TM cell [Ca2ϩ] mobilization assay, in this study and else- 11. Griffin BW, Klimko P, Crider JY, Sharif NA. AL-8810: a novel i prostaglandin F analog with selective antagonist effects at the where.15 Similar high potency and efficacy observations for 2␣ prostaglandin F (FP) receptor. J Pharmacol Exp Ther. 1999;290: travoprost acid have also been made in other cell types.14,15,19 2␣ 1278–1284. Although the reasons for the higher potency and efficacy of 12. Clark AF, Wilson K, McCartney MD, Miggans ST, Ku M, Howe W. travoprost acid (relative to unoprostone (acid) and bimatoprost Glucocorticoid-induced formation of cross-linked actin networks (amide)) are not fully understood, they may be related to its 1,7 in cultured human trabecular meshwork cells. Invest Ophthalmol high FP receptor selectivity and also may be because it is the Vis Sci. 1994;35:281–294. ϩ more potent ( )-enantiomer of racemic fluprostenol. These 13. Griffin BW, Williams GW, Crider JY, Sharif NA. FP prostaglandin attributes of travoprost acid may permit the formation of a receptors mediating inositol phosphates generation and calcium more stable and better-coupled ligand-receptor complex that mobilization in Swiss 3T3 cells: a pharmacological study. J Phar- favors a greater and faster recruitment of the appropriate G macol Exp Ther. 1997;281:845–854. proteins and phospholipase C, thus resulting in an overall 14. Sharif NA, Xu SX, Williams GW, Crider JY, Griffin BW, Davis TL. greater activation of the signal-transduction processes associ- 3 3 Pharmacology of [ H]/[ H] and ated with the FP-receptor. However, additional work is needed 3 [ H]prostaglandin F2␣ binding to EP3, and FP prostaglandin recep- to explore these possibilities further. tor binding sites in bovine corpus luteum: characterization and In conclusion, we determined the pharmacologic proper- correlation with functional data. J Pharmacol Exp Ther. 1998;286: ties of the endogenously expressed FP receptor present in 1094–1102. h-TM cells of several human donors, by using numerous syn- 15. Sharif NA, Williams GW, Kelly CR. Bimatoprost and its free acid are thetic prostaglandin FP receptor agonist analogues and an prostaglandin FP receptor agonists. Eur J Pharmacol. 2001;432: FP-receptor–selective antagonist. In addition, we demonstrated 211–213. that bimatoprost, unoprostone isopropyl ester, and their re- 16. Griffin BW, Magnino PE, Pang I-H, Sharif NA. Pharmacological spective free acids are agonists at the h-TM FP receptor where characterization of an FP on rat vascular they readily induce the generation of [3H]-IPs and rapidly and smooth muscle cells (A7r5) coupled to phosphoinositide turnover 2ϩ and intracellular calcium mobilization. J Pharmacol Exp Ther. directly mobilize [Ca ]i through the FP prostaglandin recep- tor. 1998;286:411–418. 17. Sharif NA, Wiernas TK, Howe WE, Griffin BG, Offord EA, Pfeifer Acknowledgments AMA. Human corneal epithelial cell functional responses to inflam- matory agents and their antagonists. Invest Ophthalmol Vis Sci. The authors thank Mari Engler for the initial cultures of h-TM cells, 1998;39:2562–2571. colleagues in the Medicinal Chemistry Unit for synthesis of certain PG 18. Coleman RA, Smith WL, Narumiya S. VIII. International Union of analogues used in the current studies, and Tom Dean and Mark Hell- Pharmacology classification of receptors: Properties, berg for helpful comments on the manuscript. distribution, and structure of the receptors and their subtypes. Pharmacol Rev. 1994;46:205–229. References 19. Sharif NA, Kelly CR, Crider JY. Agonist activity of bimatoprost, travoprost, latanoprost, unoprostone isopropyl ester and other 1. Hellberg MR., Sallee V, McLaughlin M, et al. Preclinical efficacy of prostaglandin analogs at the cloned human ciliary body FP pros- travoprost, a potent and selective FP prostaglandin receptor ago- taglandin receptor. J Ocular Pharmacol Ther. 2002;18:313–324. nist. J Ocul Pharmacol Ther. 2001;17:421–432. 20. Ushikubi F, Hirata M., Narumiya S. Molecular biology of prostanoid 2. Stjernschantz J, Selen G, Sjoquist B, Resul B. Preclinical pharma- receptors: an overview. J Lipid Mediat Cell Signal. 1995;12:343– cology of latanoprost, a phenyl-substituted PGF analogue. Adv 2␣ 359. Prostaglandin Thrombox Leukotr Res. 1995;23:513–518. 21. Kamphuis W, Schneemann A, van Beek LM, Smit AB, Hoyng PFJ, 3. Taniguchi T, Haque MSR, Sugiyama K, Hori N, Kitazawa Y. Ocular hypotensive mechanism of topical isopropyl unoprostone, a novel Koya E. Prostanoid receptor gene expression profile in human prostaglandin metabolite-related drug, in rabbit. J Ocul Pharma- trabecular meshwork: a quantitative real-time PCR approach. In- col. 1996;12:489–498. vest Ophthalmol Vis Res. 2001;42:3209–3215. 4. Bito LZ. Prostaglandins: a new approach to glaucoma management 22. Polansky JR, Krtz RM, Alvarado JA, Weinreb RN, Mitchell MD. with a new, intriguing side effect. Surv Ophthalmol. 1997; production and glucocorticoid regulatory mechanisms 41(suppl 22):S1–S14. in cultured human trabecular meshwork cells. Prog Clin Biol Res. 5. Woodward DF, Krauss AH-P, Chen J, et al. The pharmacology of 1989;312:113–138. bimatoprost (Lumigan௢). Surv Ophthalmol (Suppl). 2001;45: 23. Yousufzai SYL, Ye Z, Abdel-Latif A. Prostaglandin F2␣ and its ana- S337–S345. logs induce release of endogenous prostaglandins in and ciliary 6. Mukhopadhyay P, Geoghegan TE, Patil RV, Bhattacherjee P, Pater- muscles isolated from cat and other mammalian species. Exp Eye

son CA. Detection of EP2,EP4 and FP receptors in human ciliary Res. 1996;63:305–310.

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24. Dijkstra BG, Schneemann A., Hoyng PF. Flow after prostaglandin 27. Bhattacherjee P, Paterson CA, Percicot C. Studies on receptor E1 is mediated by receptor-coupled adenylyl cyclase in human binding and signal transduction pathways of unoprostone isopro- anterior segments. Invest Ophthalmol Vis Sci. 1991;40:2622– pyl ester. J Ocul Pharmacol Ther. 2001;17:433–441. 2626. 28. Thieme H, Stumpff F, Ottlecz A, Pericicot C, Lambrou GN, Wied- 25. Schachtsschabel U, Lindsey JD, Weinrib RN. The mechanism of erholt M. Mechanism of action of unoprostone on trabecular action of prostaglandins on uveoscleral outflow. Curr Opin Oph- meshwork contractility. Invest Ophthalmol Vis Sci. 2001;42:3193– thalmol. 2000;11:112–115. 3201. 26. Goh Y, Hirono S, Yoshimura K. Ocular hypotensive and adverse 29. Sharif NA, Ke T-L, Haggard K, et al. Bimatoprost hydrolysis to

effects after topical application of prostaglandin analogue, S-1033, 17-phenyl-trinor PGF2␣ by human and rabbit ocular tissues and in animals: a comparative study with UF-021 and PHXA34. Jpn J agonist activity of bimatoprost and 17-phenyl-trinor PGF2␣. ARVO Ophthalmol. 1994;38:215–227. Abstract #4080, 2002.

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