Ocular Hypotensive Activity of a Non-Peptide Bradykinin B 2-Receptor Antagonist ( WIN-64338 ) In Dutch-Belt Rabbits-A Case of Poly-Pharmacology in Action

C l i n M e d International Library Citation: Sharif NA, Klekar L, Li L, Xu S (2015) Ocular Hypotensive Activity of a NonPeptide Bradykinin B2-Receptor Antagonist (WIN-64338) In Dutch-Belt RabbitsA Case of Poly-Pharmacology in Action . Int J Ophthalmol Clin Res 2:031 Received: April 17, 2015: Accepted: August 07, 2015: Published: August 11, 2015 Copyright: © 2015 Sharif NA. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Sharif et al. Int J Ophthalmol Clin Res 2015, 2:4 ISSN: 2378-346X


Introduction
Ocular hypertension is a well-recognized and treatable biomarker that is believed to be involved in the etiology of primary open-angle glaucoma, the second leading cause of blindness in the world [1][2][3][4].This retinopathy is a painless disease that is slow but progressive and steals the patient's sight if left untreated [1][2][3][4].Over the last several decades, a number of pharmaceutical agents have been shown to be effective in reducing intraocular pressure (IOP) and have been approved for treatment of glaucoma and syringe loaded with 30µL vehicle or test substance suspended in the vehicle, was inserted into the vitreous, 4-5mm posterior of limbus at the superior temple quadrant of one eye of each rabbit.The needle was inserted at a 45 degree angle towards the back of the eye in order to avoid damaging the lens.The vehicle or test substance was slowly injected into the vitreous and the needle held in place for a minute after completion of the injection at which time it was slowly removed from the eye.This ensured minimal reflux of the injected liquid.IOP was determined at time-zero (after injection) and then at various time-points thereafter with an applanation pneumatonometer [17,18,20].This intravitreal (ivt) delivery of the test agents overcame the issues of poor ocular penetration of polar drugs such as WIN-64338 (Figure 1).Two other B 2 -receptor antagonists (FR-165649 and FR-173657; Figure 1) [22,23] and a B 1 -receptor antagonist (LF-23-1591, an analog of LF22-0542) [24] were also studied by the same ivt injection process in the Dutch-belt rabbits for comparison purposes.
In addition to the ivt administration of test agents, compounds of interest were also tested for IOP-lowering activity when instilled to conscious Dutch-belted rabbit eyes via the topical ocular (t.o.) route of administration (30µl drop) prepared in the same vehicle formulation as for ivt studies [25,26].IOP was measured with an applanation pneumatonometer after light corneal anesthesia with 0.1% proparacaine [17,18,20,25,26].After baseline IOP measurements were taken, one eye of each of 7-10 (or both eyes of 5) rabbits per compound dose was topically dosed with compound.Either the contralateral eye was dosed with vehicle or a separate group of rabbits was used for vehicle control.Subsequent IOP measurements were made at various intervals.

H]-BK and [ 3 H]-des-Arg 10 -kallidin receptor binding and other ligand binding assays
In order to verify the affinity of the key test compounds of interest (used in the above studies) for the two major BK-receptor subtypes, B 1 -receptors and B 2 -receptors, specific radio-ligand binding studies directed at these sub-types were conducted.Cell membranes of Chinese hamster ovary cells transfected with human cloned BK B 1receptors or B 2 -receptors (Euroscreen [Gosselies, Belgium]; Cerep Inc. [Le Bois L'Evegue, France]; Chantest Corp., Bethesda, MD, for custom expansion of cell cultures and cell homogenates) were used in the receptor binding assays.Briefly, cell membranes were incubated with either 0.35nM [ 3 H]-des-Arg 10 -kallidin [24] or 0.1-0.2nM[ 3 H]-BK [27] (Perkin Elmer Corp., Cambridge, MA) in the absence or presence of unlabeled Des-Arg 9 [Leu 8 ]bradykinin or BK (1µM) in 96-well microtiter plates at 23 °C to radiolabel B 1 -receptors and B 2receptors, respectively.Aliquots (50µL) of test agents or incubation buffer were added to various wells containing a total volume of 0.5mL incubation medium.The assay was continued for 1 hr and then the contents of the wells were harvested over GF/B glass fiber filters previously soaked in 0.5% polyethyleneimine under rapid vacuum (Tomtec Inc.; Gaithersburg, MD).The filter-bound radioactivity (Des-Arg 9 [Leu 8 ]bradykinin or [ 3 H]-BK) was determined by liquid scintillation spectrometry and the data analyzed using a sigmoidalfit, iterative curve-fitting computer program (ActivityBase ® ; IDBS, Surrey, UK) as previously described [17,20,26].Origin Software ® (Microcal Inc; Northampton, MD) was utilized to graph the data as necessary.All cumulative data were represented as mean ± SEM from the number of experiments conducted.
In order to determine the potential for (S)-WIN-64338 to bind to ligand binding sites other than those associated with the BK B 2 -receptors, this compound was profiled for its ability to displace radiolabeled non-BK receptors (cell surface and intracellular), non-BK-related ligand binding sites, such as transmitter uptake sites, enzymes, ionchannels and immunological factors using 1nM, 100nM and 10µM final concentrations of (S)-WIN-64338.These studies were performed at Caliper LifeSciences [NovaScreen] (Hanover, MD) employing well document assays and procedures as previously described [28] and as can be found on the following websites for PerkinElmer, Euroscreen and Cerep: http://www.perkinelmer.com/services/contractresearch/default.xhtml; http://www.euroscreen.com;http://www.cerep.fr/Cerep/Users/

Ethical statement/institutional review and approval of studies
All animal studies reported herein were reviewed and approved by the Alcon animal studies review board which also approved all the study protocols utilized in the current studies as per Alcon procedures (e.g.PROC-002851 titled "Routes of Drug Administration").Additional experimental guidelines were followed as described in the other Alcon procedural documents (PROC-0000123, PROC-0000071, PROC-0000067, and PROC-0002809).Guidelines for use of animals in research were also adopted as necessary per Association for Research in Vision and Ophthalmology (ARVO).
For ivt studies, half of a 30G1/2-inch needle, on a Hamilton

Assays to measure intracellular Ca 2+ ([Ca 2+ ] i ) mobilization
Briefly, [Ca 2+ ] i mobilization assays were conducted as previously described in detail [20,26,29] using a Fluorescence Imaging Plate Reader (FLIPR-Tetra) in conjunction with a proprietary Ca 2+ -sensitive dye (FLIPR Calcium Assay Kit) (Molecular Devices, Menlo Park, CA; http://www.moleculardevices.com/Products/Assay-Kits/GPCRs/FLIPR-Calcium.html).Isolated primary human ciliary muscle (h-CM) cells [30], trabecular meshwork cells (h-TM, both normal and glaucomatous) [31][32][33][34], and immortalized non-pigmented ciliary epithelial (ih-NPE) cells [35,36] of low passage number were grown to 80-90% confluency in 96-well microtiter plates (with bottom surface black).On the day of the experiment, the cells were loaded with the dye for an hour at 23 °C and the plates loaded into FLIPR.Aliquots (50µL) of test agent solutions or buffer were added to a total volume of 0.5mL/well and the change in fluorescence monitored over 2 min.BK (1µM) was used as an internal positive control agent, and the responses of the test drugs were represented as a percentage of the response induced by BK (set at 100%).When the antagonist potency was investigated, the compound (various concentrations) was added to the cells 20 min before challenging with the agonist, BK.Concentration-response/inhibition data were graphed and equilibrium inhibition constant (K i ) values calculated for each antagonist as previously described and references therein [20,27,29,34].All data were collated and mean ± SEM determined for each compound.

Measurement of secreted prostaglandins (PGs)
Primary normal h-TM and h-CM cells isolated from human donor eyes (see above) and ih-NPE cells were distributed in 96-well microtiter plates, grown to ~95% confluency and then exposed to various concentrations of BK, buffer or test compounds.The incubation was conducted for 1 hr at 37 °C and then terminated by removal of some of the assay incubation buffer (0.5mL) which was stored at -80 °C.For the determination of the total PGs released into the extracellular medium, the cell-incubation buffer was thawed slowly at 23 °C and then used in a competitive enzyme immunoassay kit (Cayman Chemical Co, Ann Arbor, MI) assay conducted per the manufacturer's instructions [17,18,20,34].The limits of detection for the total PGs assay were 29pg/mL.

Results
Topical ocular dosing of either enantiomer of WIN-64338 failed to modulate IOP in Dutch-belted rabbits (Table 1).Similar lack of  ).After taking baseline IOP readings, one eye of each animal was injected with 30µl aliquot of each of the afore-mentioned vehicle or drugs, and the IOP measured at various times thereafter.The mean ± SEM of the IOPs from 7-10 rabbits/treatment group are shown.All observations for both enantiomers of WIN-64338 at 8 hr postdose till 72 hr post-dose were statistically significant at levels of p<0.05-0.01,all relative to the vehicle.Note that the x-axis is not to scale but depicts the times at which the IOPs were measured after the ivt injections.However, when (S)-or (R)-WIN-64338 (50μg [0.16%]) were delivered ivt in Dutch-belted rabbits, a pronounced IOP reduction and a long duration of action was observed with both compounds (Figure 2, Table 2).Interestingly, two other non-peptide B 2 -receptorselective antagonists, FR-165649 and FR-173657, and a non-peptide B 1 -receptor-selective antagonist (LF23-1591) were essentially devoid of any ocular hypotensive activity when also tested ivt in the Dutchbelted rabbits (Table 2).In an additional study, even 10μg [0.033%] of (S)-WIN-64338 ivt was quite effective in lowering IOP , thus IOP reductions of 19.5% at 8 and 24 hrs, 32.5% at 30 hrs, 36% at 48 hrs, and 27.7% at 72 hrs IVT post-dosing were observed.(R)-WIN-64338 was also quite a potent and efficacious ocular hypotensive agent (Figure 2).BK (50μg [0.16%] IVT; used as a positive control agent) reduced IOP as follows: 22.9% at 5 hrs, 37.0% at 8 hrs, and 8.3% at 24 hrs ivt post-dosing.
In [Ca 2+ ] i mobilization assays using isolated primary hCM cells, (S)-WIN-64338 caused 17-19% and (R)-WIN-64338 caused 13% increase in [Ca 2+ ] i mobilization at 10μM, behaving as weak partial agonists.Similarly, both (S)-and (R)-enantiomers of WIN-64338 (10μM) induced a small but significant amount of total PGs production and secretion from hTM cells (30 ± 1 and 31 ± 2% relative to 100% caused by 1μM BK) and from h-CM cells (126 ± 107% and 163 ± 11% relative to 100% caused by 1μM BK).Lower concentration of these enantiomers increased total PGs release from h-TM and h-CM cells to a lesser degree than BK (Figure 2A-C).For reference, 1μM BK simulated secretion of 571pg/ml total PGs from h-TM cells, while 171pg/ml total PGs were secreted from h-CM cells (detection limit was 29pg/ml).However, when tested as an antagonist against 1μM BK in the [Ca 2+ ] i mobilization assays under non-equilibrium conditions in normal primary isolated h-CM, h-TM cells and in ih-NPE cells, WIN-64338 exhibited relatively weak antagonist properties (K i =157-425nM), as compared to HOE-140 (K i =0.8-7nM) and other nonpeptide B 2 -receoptor antagonists (Table 2).

Discussion
Recent animal studies have documented that the peptide B 2receptor agonist, BK, but not two different the B 1 -receptor agonists, causes robust ocular hypotension in Dutch-belt rabbits injected ivt, and increases outflow facility in perfused bovine eye anterior chambers [20,34].Furthermore, t.o.dosing of two different nonpeptide B 2 -receptor-agonist synthetic small molecules (FR-190997 and BKA278) [17,19] also induces a strong IOP-lowering response in ocular hypertensive eyes of cynomolgus monkeys that was completely blocked by a B 2 -receptor-antagonist.These collective data clearly indicated that stimulation, not blockade, of the B 2 -receptor is necessary for the observed reduction of IOP in the rabbits and monkeys.Thus, the remarkable magnitude and long duration of IOP-lowering induced by ivt injected enantiomers of WIN-64338 [21,37], a well-known B 2 -receptor-antagonist, is unexampled to our knowledge.This apparently discrepant, yet novel, observation was quite specific for WIN-64338 since two other non-peptide B 2receptor-antagonists (FR-165649 and FR-173657) [22,23] essentially lacked ocular hypotensive activity in the same animal model when the compounds were also delivered ivt and at the same dose as the (S)-and (R)-WIN-64-338 [21,37].These data necessitated additional studies to help delineate the mechanism(s) involved in the WIN-64338-induced IOP reduction.
First we needed an explanation for the observation that t.o.administration of (R)-WIN-64338 in rabbits (Table 1) was ineffective in causing IOP-lowering whereas ivt injection of the same compound caused up to 50% reduction of IOP compared with the vehicle (Figure 2).An examination of the structure of WIN-64338 shows that the molecule is very polar due to the phosphonium moiety and the hydrochloride salt (Figure 1), thus rendering the drug relatively inaccessible to the anterior chamber upon t.o.dosing.Clearly, delivering the compound directly into the back of the eye by ivt injection overcomes this hurdle, although there's a delay in its action due to the diffusion needed from the vitreous to the ciliary body and trabecular meshwork (Figure 2).The fact that the (R)-enantiomer was approx.twice as potent as the (S)-enantiomer of WIN-64338 in causing ocular hypotension in the rabbits (Figure 2) indicated a stereoisomeric distinction commensurate with the slightly different functional activity of the two enantiomers.The rather slow onset of action of both enantiomers of WIN-64338 in the rabbit IOP model may be related to the fact that generation and release of other endogenous transmitters or other substances was necessary [37].Indeed, it seems that the small amount of [Ca 2+ ] i mobilized by WIN-64338 (see Results) followed by the induction of PG synthase and the subsequent synthesis and secretion of PGs (Figure 3A-C) are part of the reason.However, the high magnitude of the IOP-lowering response induced by both (R)-and (S)-WIN-64338 may result from recruitment of multiple receptors and their signaling pathways, including adrenergic, dopaminergic, muscarinic receptors, ion-channels, transporters, etc as revealed by the ligand binding profile of (S)-WIN-64338 (Table 3).
The opioid receptor binding activity of (S)-WIN-64338 (Table 3) may have some significance to its ability to lower IOP in the rabbits as have been demonstrated for kappa-opioid agonist bremazocine [50][51][52] and for mu-opioid agonist morphine [53].These compounds modulate norepinephrine release and cause generation of nitric oxide and carbon monoxide [52,53], which then raise intracellular cGMP to cause ocular hypotension.Similarly, (S)-WIN-64338 has a relatively high affinity for sigma receptors [54], and ion-channels [54][55][56] for Na + , K + and Ca 2+ (Table 3).Since sigma agonists [54], and inhibitors of the latter channels have some IOP-lowering activities, it is likely that ocular hypotension caused by WIN-64338 may have some contribution from this receptor and ion-channels.
Unlike the linkage of many ligand binding sites, receptors and ion-channels to IOP reduction discussed above, the direct interaction of (S)-WIN-64338 with protein kinase C, and with receptors for cholecystokinin, neurokinin (substance P), neuropeptide Y and somatostatin (Table 3) currently does not appear have any correlative evidence available in the literature.However, since these peptides are present in ocular tissues and/or in the aqueous humor [57][58][59][60], further studies appear warranted to investigate the role of receptors of these agents in modulation of IOP.
A fascinating area connected with the multitude of affinities and functional activities of both (R)-and (S)-WIN-64338 is the role that poly-pharmacology plays in mediating reduction of elevated IOP.Pharmacological selectivity for specific receptor or enzyme targets is relative to the number of on-target and off-target activities of the compounds.Furthermore, it is the actual concentration achieved at the target protein that determines the engagement of that entity in down-stream effects of the compound in terms of activating or inhibiting the signaling pathway(s) at the cellular level.This is further complicated by the agonist/partial agonist/ inverse agonists/ antagonist nature of the compounds for receptors, and activators/ inhibitors for the enzymes and ion-channels.It is well known that until the discovery and use of modern pharmacological tools, delineation of receptor sub-types and enzyme sub-classes was not possible.Thus, in the current context several compounds with mixed pharmacological activities have been identified and shown to lower IOP in various animal models of ocular hypertension.For example, flesinoxan (5HT 1A agonist/alpha-1 adrenoceptor-antagonist) [42], flunarizine (sigma ligand and Ca 2+ -channel blocker) [54,55], cabergoline (dopamine and serotonin receptor agonist) [26,40], sulprostone (EP 1 and EP 3 prostaglandin [PG] agonist) [60], AL-6598 (DP and EP 2 PG receptor agonist) [61,62], and unoprostone (FP receptor agonist/K +channel blocker) [63] produce robust IOP reduction probably due to their multiple pharmacological activities.Therefore, the varied and numerous receptor/transporter and ion-channel activities associated with WIN-64338 (in addition to its first described BK B 2receptor antagonist actions; Table 3) are most likely the contributors to its profound ocular hypotensive actions in the rabbits observed in the current studies (Table 2, Figure 2).Such poly-pharmacology may be very useful since it is precisely the idea behind combination products [13,14,64,65] where differing pharmacological classes of compounds, with different mechanisms of action, are formulated together to provide fixed-dose combination ocular hypertensive  or triple combinations (e.g.dorzolamide + brimonidine + timolol) [13,14,64,65].
In addition to the above poly-pharmacology, there's a distinct possibility that depending on the cells, tissues, and animal species being utilized in the in vitro and in vivo studies, WIN-64338 may exhibit partial agonist or antagonist properties.Thus, in the current studies we observed a small (13-19% above baseline) partial agonist effect of (S)-WIN-64338 on [Ca 2+ ] i mobilization in human ocular cells involved in aqueous humor regulation (h-CM and h-TM cells).Similarly, a 30% and 126-163% increase in PGs release from h-TM and h-CM cells, respectively, was observed when exposed to WIN-64338.Such mixed pharmacological actions of WIN-64338 are not unprecedented since another BK B 2 -receptor antagonist, HOE-140, has behaved as an agonist and antagonist in isolated blood vessels of different species [66], and is a potent mitogenic agonist in a number of cell-lines [67], despite displaying classical antagonist activity in numerous other biological systems [68][69][70].Moreover, there's the possibility that WIN-64338 is an activating ligand for a new subtype of BK receptor, as has been proposed from functional studies conducted in other tissues and species [71][72][73][74][75].
The relatively small increase in [Ca 2+ ] i mobilization and PGs secretion induced by (S/R)-WIN-64338 may be considered inadequate to affect IOP.However, due to signal amplification and recruitment of additional down-stream signal transduction processes associated with receptor activation, especially PG receptors [76] that involves matrix metalloproteinase (MMP) release [77], the amount of PGs released by (S/R)-WIN-64338 are more than sufficient to trigger various PG receptors in target cells involved in AQH regulation [20,32,33,36].Thus, using the data from figure 3A-3C (also see Results), it becomes apparent that upon secretion h-TM and h-CM cells into the extracellular medium, concentrations of 0.15-0.21nMPGs can be achieved.Since PGs are very potent agents with halfmaximal receptor activation of EP 3 receptors at 0.41-0.63nM(by PGE 2 and PGF 2α ) [78], and of EP 4 receptors at 0.17-0.22nM(by PGE 1 and PGE 2 ) [78][79][80], for instance, appreciable receptor activation would be expected to occur that would result in generation of MMPs [77] that in turn degrade extracellular matrix to lower IOP.

Conclusions
In conclusion, the effective IOP-lowering activity of both enantiomers of WIN-64338 observed in Dutch-belt rabbits after ivt injections is indicated to be due to the poly-pharmacology and mixed agonist/antagonist properties associated with this compound.Discovery of other compounds that may provide such robust ocular hypotension is eagerly awaited.

Figure 2 :
Figure 2: Changes in Dutch-belt rabbit IOPs following ivt injection of vehicle, or (R)-WIN-64338 (20µg [o.066%]) or (S)-WIN-64338 (50µg [0.16%]).After taking baseline IOP readings, one eye of each animal was injected with 30µl aliquot of each of the afore-mentioned vehicle or drugs, and the IOP measured at various times thereafter.The mean ± SEM of the IOPs from 7-10 rabbits/treatment group are shown.All observations for both enantiomers of WIN-64338 at 8 hr postdose till 72 hr post-dose were statistically significant at levels of p<0.05-0.01,all relative to the vehicle.Note that the x-axis is not to scale but depicts the times at which the IOPs were measured after the ivt injections.

Figure 3 :
Figure 3: Total prostaglandins released by different concentrations of (S)-WIN-64338 or (R)-WIN-64338 from isolated primary, normal h-TM and h-CM (C) cells are shown.The values on the y-axes are PGs secreted (A,B) into the extracellular medium of the cells in culture plates in response to the drugs as a % of the response induced by the natural agonist of B 2 -receptors, BK (1µM), the response for which was set at 100%.Only the responses induced at 1µM and 3µM of each enantiomer of WIN-64338 were statistically significant (p<0.05)relative to basal levels of PGs.

Table 1 :
Effect of topical ocular (R)-WIN-64338 on Dutch-belt rabbit IOP Compound Time

Table 2 :
Effect of IVT administration of various non-peptide B 1 -and B 2 -receptor antagonists on Dutch-belt rabbit IOP nd Data are mean ± SEM from 7-10 rabbits per group.The mean baseline IOP of these animals ranged from 27.5 ± 0.4 to 28.4 ± 0.4mmHg during the course of these studies.The vehicle had minimal effect on IOP as shown in Figure2.No effects on IOP of (S)-WIN-64338 ivt were observed in the contralateral eyes.The receptor antagonist potencies refer to the non-equilibrium blocking effects of the antagonists of BK-induced [Ca 2+ ] i mobilization in isolated primary h-TM, h-CM and ih-NPE cells to illustrate the differences in relative affinities of the compounds for their respective BK-receptor sub-type(s), primarily the B 2 -receptor.*p<0.01;** p<0.001 relative to vehicle baseline IOP or relative to IOP at 4 h post-dose.nddenotesnot determined.IOP-lowering data for 10µg (0.033%) ivt injection of (S)-WIN-64338 are shown in the Results section.ISSN: 2378-346XSharif et al.Int J Ophthalmol Clin Res 2015, 2:4

Table 3 :
Ligand binding profile of (S)-WIN-64338 at numerous receptors, enzymes, transporters and ion-channels Each value is the average of two determinations for each concentration of (S)-WIN-64338 (1nM, 100nM and 10µM final).The bolded values meet the criterion of at least 50% inhibition of ligand binding for at least one concentration of the test compound.The negative value suggests an apparent stimulation of binding.There is a ± 15% variability associated with each ligand binding assay.