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Physiology and Pharmacology Myopia-Inhibiting Concentrations of Muscarinic Receptor Antagonists Block Activation of Alpha2A-Adrenoceptors In Vitro

Brittany J. Carr,1–4 Koichiro Mihara,3,5 Rithwik Ramachandran,3,5,6 Mahmoud Saifeddine,3,5 Neil M. Nathanson,7 William K. Stell,1,2,8 and Morley D. Hollenberg3,5,9 1Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada 2Alberta Children’s Hospital Research Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada 3Inflammation Research Network-Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada 4Department of Neuroscience, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada 5Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada 6Department of Physiology and Pharmacology, Western University, London, Ontario, Canada 7Department of Pharmacology, University of Washington, Seattle, Washington, United States 8Department of Cell Biology and Anatomy, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada 9Department of Medicine, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada

Correspondence: William K. Stell, PURPOSE. Myopia is a refractive disorder that degrades vision. It can be treated with , a Health Sciences Centre B101, 3330 muscarinic receptor (mAChR) antagonist, but the mechanism is unknown. Hospital Drive NW, Calgary, Alberta, Atropine may block a-adrenoceptors at concentrations ‡0.1 mM, and another potent myopia- Canada, T2N 4N1; inhibiting ligand, toxin-3 (MT3), binds equally well to human mAChR M4 and a1A- and [email protected]. a -adrenoceptors. We hypothesized that mAChR antagonists could inhibit myopia via a - Brittany J. Carr, Eye Care Centre 2A 2A Room 330, 2550 Willow Street, adrenoceptors, rather than mAChR M4. Vancouver, British Columbia, Cana- METHODS. Human mAChR M4 (M4), chicken mAChR M4 (cM4), or human a2A-adrenergic da, V5Z 3N9; receptor (hADRA2A) clones were cotransfected with CRE/promoter-luciferase (CRE-Luc; [email protected]. agonist-induced luminescence) and Renilla luciferase (RLuc; normalizing control) into human Submitted: July 7, 2017 cells. Inhibition of normalized agonist-induced luminescence by antagonists (ATR: atropine; Accepted: April 24, 2018 MT3; HIM: ; PRZ: ; TRP: ; OXY: oxyphenonium; QNB: 3- Citation: Carr BJ, Mihara K, Rama- quinuclidinyl benzilate; DIC: dicyclomine; MEP: ) was measured using the Dual- chandran R, et al. Myopia-inhibiting Glo Luciferase Assay System. concentrations of muscarinic receptor RESULTS. Relative inhibitory potencies of mAChR antagonists at mAChR M /cM , from most to antagonists block activation of al- 4 4 least potent, were QNB > OXY ‡ ATR > MEP > HIM > DIC > PRZ > TRP. MT3 was 563 less pha2A-adrenoceptors in vitro. Invest Ophthalmol Vis Sci. 2018;59:2778– potent at cM4 than at M4. Relative potencies of mAChR antagonists at hADRA2A, from most to 2791. https://doi.org/10.1167/ least potent, were MT3 > HIM > ATR > OXY > PRZ > TRP > QNB > MEP; DIC did not iovs.17-22562 antagonize.

CONCLUSIONS. Muscarinic antagonists block hADRA2A signaling at concentrations comparable to those used to inhibit chick myopia (‡0.1 mM) in vivo. Relative potencies at hADRA2A, but not M4/cM4, correlate with reported abilities to inhibit chick form-deprivation myopia. mAChR antagonists might inhibit myopia via a2-adrenoceptors, instead of through the mAChR M4/cM4 receptor subtype. Keywords: atropine, form-deprivation myopia, nonspecific binding, off-target, , alpha2a-adrenoceptor

yopia (near- or short-sightedness) is a common eye and upon cessation, ‘‘rebound’’ may occur, wherein myopia M condition that increases the risk of ocular pathology as returns at a faster rate than in untreated eyes6,7—possibly it progresses.1,2 As its prevalence continues to rise,3 so does the because of the desensitization of target receptors in response to need for an effective therapy. Topical atropine is effective a high concentration of drug. Recent studies against myopia in children4; however, treatment requires daily have supported the use of 0.01% atropine to treat childhood application, and the dosage most commonly prescribed for myopia.7,8 This comparatively lower dose of atropine appears many years (1%) may induce allergic reactions, as well as to retain its effectiveness for inhibiting myopia, and results in a muscarinic (mAChR) M3-mediated side reduced severity of side effects such as allergies, loss of effects—mydriasis, photophobia, cycloplegia—and possibly accommodation, and rebound. Some of those treated with early presbyopia.4,5 Atropine administered topically at concen- 0.01% atropine still complain of muscarinic receptor (mAChR) trations of 1% loses effectiveness over a period of 1 to 2 years; blockade-induced complications, including photophobia and

Copyright 2018 The Authors iovs.arvojournals.org j ISSN: 1552-5783 2778

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blurred vision; however, those symptoms are not severe chick eyecups comprising the RPE–choroid–sclera, it did so enough to prompt a discontinuation of treatment. Therefore, only at high concentration (5 mM); other mAChR-targeted 0.01% atropine is currently the most favored concentration antagonists, such as oxyphenonium (1 mM) and dicyclomine prescribed by clinicians both in Southeast Asia and North (0.6 mM), had no effect on the choroid in these preparations.27 America.9–12 Unfortunately, the effect of atropine on choroidal thickness in Atropine is a potent nonselective mAChR antagonist. Thus, chick eyecups lacking retina was not tested. it has been widely assumed to inhibit myopia via blockade of Of particular interest in the above examples is the mAChRs. Another potent mAChR antagonist, mamba toxin-3 requirement of high concentrations of atropine and other (MT3), is strongly selective for mAChR M4 over other mAChR mAChR antagonists for myopia-inhibiting effects, relative to the 13 subtypes in mammals, and inhibits form-deprivation myopia subnanomolar affinities (Ki) of many of these antagonists for (FDM) in the chick at far lower concentrations than those the M4 mAChR (Table 1). Indeed, even if a drug binds required for atropine (17.5–70 pmol versus 20–2000 nmol per selectively to its preferred receptors with high affinity (e.g., Ki injection, respectively).14–16 Therefore, given the ability of 1 nM), it is expected that at concentrations two to three MT3 to block myopia development in the chick model, it has orders of magnitude higher it will bind to, and affect the 21 been suggested that the mAChR M4 receptor subtype is the activity of, other (‘‘off-target’’) receptors. Thus, atropine, target for myopia-controlling action of both atropine and while receptor-selective at nanomolar concentrations, would MT3.14,17 It is important to point out, however, that a direct very likely have off-target effects at other receptors at the mechanistic link between mAChR systems in the eye and the micro- to millimolar concentrations used to mitigate myopia. mAChR antagonist properties of atropine or MT3 for inhibition Additionally, while it is true that MT3 can inhibit myopia at of myopia has never been demonstrated. much lower concentrations than atropine,14,15 this result Moreover, some evidence suggests that the ability of cannot be interpreted to mean that it does so through M4 or atropine to inhibit myopia development may not involve any other mAChR on this basis alone. Although MT3 is 18 13,28 mAChRs at all. For example, most mAChR antagonists, even selective for the M4 receptor over other mAChRs, it has a very potent ones such as 3-quinuclidinyl benzilate (QNB) or comparably high inhibitory potency (IC50 ¼ 1–10 nM) at dicyclomine, do not inhibit myopia in chicks, and those human a1A-, a1D-, and a2A-adrenergic receptors; moderate muscarinic antagonists that do so inhibit myopia require inhibitory potency (IC50 ¼ 25–50 nM) at a1B-anda2C- concentrations that can be orders of magnitude above their adrenergic receptors; and low inhibitory potency (IC50 ¼ 200 16,19 29,13 inhibition constants for blocking mAChRs (Table 1). nM) at the mAChR M1 receptor. There are also data to Furthermore, ablating ‡90% of acetyltransferase indicate that at high concentrations (1–100 lM) atropine can (ChAT)-containing amacrine cells—the only known source of also have antagonist activity at a-adrenoceptors,30,31 although retinal acetylcholine (ACh; the natural agonist at mAChRs)—in the specific adrenoceptor subtypes to which it may bind the chick has no effect on the eyes’ ability to achieve remain unknown. emmetropia, nor does elimination of retinal ChAT impair Chicks are a popular animal model for myopia drug studies. myopia inhibition by atropine.20 Treatment with myopia- They have high-quality vision, active accommodation and inhibiting concentrations of atropine in chick retina–RPE– emmetropization, and large eyes for easy intravitreal drug choroid–sclera preparations causes a massive, nonspecific delivery. Diffusers32–34 and negative lenses35,36 induce large release of retinal neurotransmitters (including dopamine), changes in axial length and refractive error (5to15 diopter and induces spreading depression that affects the entire retina, [D]) in a matter of days, and results from chick experiments not just mAChR-related circuits.21 Finally, significant myopia- can usually be replicated in mammals and nonhuman related changes in eye size in the tree shrew and chick do not primates.37 Interpretations of results, using mAChR antagonists result in significant changes in mRNA or protein expression of to prevent FDM in the chick, have generally presumed that any mAChR subtype in the retina,22,23 nor does myopia- these ligands work the same way at avian receptors as they do induced axial elongation result in changes in the concentration at human or other mammalian receptors. That said, the chick of retinal ACh, or its metabolite choline.24 Results such as these retinal structure and G-protein receptor sequences differ have led to arguments that favor a nonretinal mechanism of significantly from those of mammals. Chickens lack an atropine-mediated myopia inhibition, with mAChR binding in orthologue for the mammalian mAChR M1. Instead, chicken the choroid or sclera suggested as the most likely alternative. M2 (cM2), the orthologue for the mammalian M2 receptor, has One way to determine whether the molecular targets of anti- some pharmacologic properties of both mammalian M1 and M2 myopia therapy—that are modified directly by treatment with receptors, such as relatively high affinity for pirenzepine.38 mAChR antagonists—are located in these tissues, is to look for Thus, in the chick, one can conclude that MT3 will target the changes in receptor density upon induction of experimental M4 mAChR and not other mAChR subtypes. Furthermore, there myopia and during atropine treatment. There is no change, are examples of substantial differences in ligand behavior however, in mAChR mRNA expression in either the choroid or between species, even for mammals, whereby even minor sclera, upon induction of experimental myopia in intact eyes of sequence differences can cause receptor-selective ligands to tree shrew and chick.22,23 A second method to determine interact differently, with the same G-protein-coupled receptor whether the choroid or sclera is directly responsible for (GPCR) orthologue.39,40 We therefore considered it likely that atropine-mediated myopia inhibition is to examine the MT3, which is highly selective for the mammalian M4 receptor, responses to atropine in these tissues specifically, in in vitro might have a significantly altered ability to bind to the chick M4 preparations of eyecups lacking retina. In such preparations, receptor. the action of atropine to inhibit extracellular matrix produc- Given the likelihood that the concentrations of atropine tion and sulfate incorporation into the glycosaminoglycans of used for treating myopia in humans and chicks can act at 25 chick scleral cartilage and the inhibition of - receptors other than mAChR M4, we focused on the molecular 26 induced mouse scleral fibroblast proliferation were observed pharmacology of the chick cM4 and human M4 receptors, and only at high concentrations of atropine (0.5–100 lM in the the interactions of these receptors with the mAChR-targeted medium), which are 500- to 10,000-fold higher than those that agents that have been used to evaluate myopia development in block mAChRs (Ki ¼ 1–10nM;Table1).Finally,while the chick model. Furthermore, since both MT3 and atropine pirenzepine treatment has been shown to result in changes can potentially interact with a-adrenoceptors, we also ex- in choroidal thickness when applied to in vitro preparations of plored the interactions of FDM-mitigating agents with the

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TABLE 1. Binding Data for Muscarinic Antagonists Tested at mAChR M4 and cM4 in Our Assay

Drug IC50 Study Ki(CP) Study Ki Literature Inhibits FDM Est. [Vit] Source Cat No.

Muscarinic antagonists at mAChR M4 and cM4 ATR *390 pM *140 pM *125–250 pM70,123 Yes16, 0.1–10 mM Sigma A0257 †710 pM †120 pM †400–630 pM38 MT3 *8 nM *3 nM *2 nM29,124 Yes14,15, 10–350 nM Peptides International PMT-4410-s †450 nM †75 nM †N/A HIM *1 nM *4 nM *8 nM125 Yes19, 3–12 mM Enzo Life Sciences ALX-550-061 †6 nM †1 nM †6 nM19 OXY *400 pM *145 pM *100–160 pM126 Yes16, 1–10 mM Sigma O5501 †560 pM †95 pM †N/A PRZ *23 nM *8 nM *16–80 nM70 Yes16, 10 mM Sigma P7412 †50 nM †8 nM †4–6 nM38 QNB *115 pM *40 pM *315 pM125 Partial16, 0.74 mM Sustainable Scientific BR-143013 †300 pM †50 pM †30–100 pM38 TRP *155 nM *55 nM *16 nM125 Partial16, 10 mM Sigma T9778 †245 nM †40 nM †63 nM127 MEP *1 nM *360 pM *N/A No16, 1–10 mM Sigma M5651 †4 nM †590 pM †N/A DIC *15 nM *5 nM *5–160 nM126 No16, 1–10 mM Sigma D7909 †40 nM †7 nM †5 nM127

Drug ED50 Study pKi(CP) Study pED50 Literature Inhibits FDM Est. [Vit] Source Cat No. CCh *5.6 lM N/A *4 lM123 N/A Tocris 2810 †2 lM †3–5 lM51

123 126 19,38,127 Non-M4/cM4-specific receptors/tissues: rat M4 ; guinea pig atria and ileum ; chick heart. IC50 Study data are the data determined by 49 our binding assay, Ki(CP) Study data are calculated from the IC50 values obtained by our assay by the method of Cheng and Prusoff, and Ki Literature data are data from previously published studies. Maximum inhibition achieved by all antagonists was 100%. ATR, atropine sulfate; OXY, ; PRZ, pirenzepine dihydrochloride; CCh, carbachol; Est. [Vit], estimated vitreal concentrations, assuming uniform dilution in total vitreal volume, required for myopia inhibition in the chick; TRP, tropicamide; N/A, not available. * Human or mammalian mAChR M4 receptor or tissue. † Avian mAChR M4 receptor or tissue.

human a2A-adrenergic receptor ADRA2A (hADRA2A) subtype. response element (CRE)-luciferase reporter construct, such To test the hypothesis that non-mAChR mechanisms can that increased luciferase activity serves as an index of mediate the inhibition of myopia development by mAChR activation of the transfected muscarinic and adrenergic antagonists in the chick FDM model, we evaluated (1) whether receptors. Carbachol was used to activate the chick and mAChR antagonists have similar relative inhibitory potencies at human mAChRs, and clonidine was used as the agonist to chick cM4 compared to human M4, and (2) whether mAChR activate hADRA2A. The ability of increasing concentrations of antagonists can bind to, and inhibit signaling by, hADRA2A. We antagonists to inhibit the and adrenergic responses chose to investigate the binding of mAChR antagonists to a2A- was used as an index of their relative potencies for blocking adrenoceptors because MT3 has a high affinity for these the expressed receptors. The relative inhibition constants of adrenergic receptors, because this adrenoceptor is the most the myopia-inhibiting compounds, for blocking either the common a-adrenoceptor subtype in the central nervous muscarinic or a2A-adrenergic receptors, were compared 41 system, and because some a2-adrenoceptor agonists have directly with the relative ability of the compounds to mitigate been reported to inhibit FDM in the chick (Carr BJ, Stell WK. myopia in the chick FDM model. IOVS 2016;57:ARVO E-Abstract 4738) and guinea pig.42 DNA and Expression Vectors

METHODS AND MATERIALS All receptor clones were expressed in a pcDNA 3.1(þ) vector (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA). The 18 Experimental Strategy cM4 genomic clone was obtained as previously described, and the M4 (MAR0400000) and hADRA2A (AR0A2A0000) Our strategy to explore the interactions of the FDM-related receptor clones were purchased from the cDNA Resource antagonists with either mAChR M4/cM4 or hADRA2A was to Center (Bloomsburg University Foundation, Bloomsburg, PA, express the recombinant receptors in a mammalian Lenti-X USA). The cAMP response element luciferase vector (CRE-Luc; HEK 293T cell line, in which the only endogenously expressed pGL4.29[luc2P/CRE/Hygro]) and the constitutively active mAChR (M3) was eliminated by a CRISPR/Cas9-mediated Renilla luciferase control vector (RLuc; pRL-RK) were pur- genomic editing approach (CRISPR-M3 cells: CR-M3). The chased from Promega (Madison, WI, USA). HEK cell line is well recognized for its ability to couple the signaling of GPCRs from multiple species efficiently to multiple CRISPR/Cas9 Knockdown of mAChR M3 effectors, including Gs,Gi,Gq, and G12/13. In this system, the abundance of expressed muscarinic and adrenergic receptors CRISPR knockout of mAChR M3 in Lenti-X HEK 293T cells enables coupling to Gs, with a resulting increase in synthesis of (LX293T; Clontech, Mountain View, CA, USA) was performed to cyclic AMP (cAMP).43,44 In turn, the cAMP drives a cAMP ensure that mAChR ligand-mediated effects on cAMP were the

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result of activity at transfected, and not endogenous, recep- was chosen, on the basis of preliminary concentration– 45,46 tors. Stable CRISPR M3-knockout LX293T cells are referred response studies, to produce large but submaximal activation to as CR-M3 cells. Cell culture medium, serum, transfection of normalized CRE-Luc luminescence (LucN). Submaximal materials, culture flasks, and multiwell plates were purchased concentrations (78%–90% activation) were used instead of from ThermoFisher Scientific unless specified otherwise. The 50% or 100% activation, so as to extend the dynamic range of knockout design and procedures used to derive the CR-M3 cells measurable responses. The choice of antagonists for testing from the wild-type LX293T cells, using the GeCKO CRISPR was based on availability and previously published reports of protocol (available in the public domain, https://www.addgene. their ability to prevent FDM in the chick (Table 1). All drug org/crispr/libraries/geckov2/), were as described by Sanjana et stock solutions (10 mM) were made using sterile distilled H2O, 47 48 al. and Shalem et al. Three sets of genome-specific single with the exception of QNB (5 mM) and himbacine (9 mM), guide RNA sequences to mAChR M3 (F to R) were chosen from which were dissolved in 100% and 90% methanol, respectively. the GeCKOv2 Human Library, and both strands of oligonucle- The maximum concentration of methanol that did not cause otides were synthesized for each target sequence (FA:CACCGgtc cell death, as determined by Robust regression and Outlier acaagcgcgcacccgagc; RA: AAACgctcgggtgcgcgcttgtgacC; FB: removal (ROUT; GraphPad, La Jolla, CA, USA) statistical CACCGgcggtaccaccgatgaccctc; RB: AAACgagggtcatcggtgg analysis, was 6%. taccgcC; FC: CACCGgcgctttcttaacgggcatcc; RC: AAACggatgcccgt Changes in intracellular cAMP were measured indirectly taagaaagcgcC); the lowercase sequences are target sequences of using the Dual-Glo Luciferase Assay System and protocol the M3 genomic locus, and the uppercase sequences are (Promega). Drug-treated cells and Dual-Glo assay reagents were flanking sequences for cloning. Both strands of the oligonucle- equilibrated to room temperature, and then 50 lL Dual-Glo otides were annealed and inserted in BsmBl restriction enzyme Luciferase Reagent was added to each sample well. The plate sites under a U6 promoter in a CRISPR/Cas9 vector (lentiCRISPR was incubated for 10 minutes at room temperature, with v2), a gift from Feng Zhang (Addgene plasmid #52961; moderate shaking to ensure complete cell lysis. After Cambridge, MA, USA). The lentiCRISPR v2 plasmids containing incubation, CRE-Luc levels were measured using a Victor X4 three M3-targeting sequences were mixed and transfected using Spectrophotometer (PerkinElmer, Waltham, MA, USA). Once Lipofectamine LTX Reagent. The transfected cells were main- CRE-Luc measurements were complete, 50 lL Dual-Glo Stop- tained in the presence of 5 mg/mL puromycin to select for and-Glo Reagent was added to each of the sample wells, the 10- knockout cells. Once established, stable-knockout CR-M3 cells minute incubation with shaking was repeated, and then the were resuspended in high-glucose Dulbecco’s modified Eagle’s levels of RLuc (normalizing control) in the wells were medium (DMEM) 10% dimethyl sulfoxide and frozen in liquid þ measured in the same order, using the same spectrophotom- nitrogen for future use. eter. Cell Culture and Transfection Protocols Data Analysis CR-M cells were grown in high-glucose DMEM supplemented 3 Raw RLuc data were first subjected to outlier detection using with 10% fetal bovine serum, 1 mM sodium pyruvate, and 5 ROUT analysis with a 1% false detection rate (GraphPad Prism v lg/mL Plasmocin (InvivoGen, San Diego, CA, USA) in a 5% CO2 environment at 378C. Cells were maintained at <80% 6.07; La Jolla, CA, USA). Flagged data usually represented confluency and passaged with phosphate-buffered isotonic significant decreases in RLuc expression, due to cell death saline, pH 7.4, containing enzyme-free EDTA (1 mM), with caused by high-concentration drug or methanol. There were mild trypsinization (90 lL 0.25% trypsin-EDTA/25 cm2 T-flask) no instances of significant cell death for any of the experiments to optimize homogeneity during replating. Transfection was involving testing at M4 or cM4. At hADRA2A, cell death performed in 12-well plates at 30% confluency. For each well occurred at dicyclomine hydrochloride (DIC) (>500 lM), QNB to be transfected, 4 lL Lipofectamine LTX was diluted in 50 lL (>300 lM, 6% methanol), oxyphenonium (OXY) (>3 mM), Opti-MEM reduced serum medium; vector cDNAs (CRE-Luc mepenzolate bromide (MEP) (>10 mM), and himbacine (HIM) [180 ng], RLuc [160 ng], and receptor genomic clones [160 (>500 lM). These data were removed along with their ng]) were mixed in a separate aliquot of 50 lL Opti-MEM. corresponding CRE-Luc values in the final analysis. Remaining Equal volumes of the LTX and DNA solutions were then CRE-Luc data were normalized to RLuc (CRE-Luc/RLuc ¼ LucN), combined (1:1 ratio), and the cells were incubated at room and then either the fold-change LucN (agonist treatment: temperature for 5 minutes, after which the LTX-DNA [treated well well without agonist] ‚ well without agonist) complexes were added (100 lL LTX-DNA complex/well) to or the percent maximum response (antagonist treatment: wells with gentle mixing. The cells were allowed to grow in [treated well ‚ well without antagonist] 3 100) was calculated. 5% CO2 at 378C, and the cell medium was changed to For graphing and IC50 calculations, molar concentrations were supplemented DMEM without selection after a minimum graphed on the x-axis, transformed using the algorithm X ¼ exposure of 8 hours to the transfection medium. Twenty-four Log(X), and then nonlinear regression analysis was performed. hours after transfection, cells were seeded (7500 cells/well) Graphical data are represented as curve-fitted linear regres- into white, clear-bottomed, tissue-treated 96-well plates, fed sions of the means of the transformed molar concentration with supplemented DMEM without selection, and allowed to values 6 SEM (GraphPad Prism, Version 6.07); n ¼ 3or4 grow overnight. experiments, performed in duplicate. Inhibitory constants (Ki(CP)) were estimated from these data by the method of 49,50 CRE-Luc Luminescence Assay and Antagonist IC Cheng and Prusoff (pKi(CP) ¼ IC50 ‚ (1 þ ([A]/EC50)), 50 where [A] agonist concentration used, and EC was the Curves ¼ 50 agonist concentration required to elicit a 50% response, as Forty-eight hours after transfection, the cell medium was determined by previous experiments using our assay system. aspirated and replaced with various concentrations of antag- Data are reported as either IC50 (assay/system-dependent onist diluted in FluoroBrite DMEM containing a fixed inhibitory potency), Ki (absolute inhibitory potency), or EC50 concentration of agonist (50 lL/well); treated cells were (assay/system-dependent excitatory potency) values. These incubated for 4 hours at 378C under 5% CO2 þ O2. The fixed values are all concentration-dependent; therefore, the smaller agonist concentration (carbachol: 10 lM; or clonidine: 1 lM) the value, the more effective the ligand.

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FIGURE 1. Inhibitory potencies of various mAChR antagonists at the human M4 receptor. Antagonists are categorized into full myopia inhibition (open/colored symbols), partial myopia inhibition (partially filled symbols), and no myopia inhibition (filled symbols) according to previously 14,16,19 published research, and ranked in descending order from most to least potent in our receptor assay (IC50 6 95% CI). MT3 (yellow) and atropine (blue) are indicated separately as they are the most researched anti-myopia mAChR antagonist in humans (atropine), and the most potent anti-myopia drug found to date in chicks (MT3). ATR, atropine sulfate; OXY, oxyphenonium bromide; PRZ, pirenzepine dihydrochloride; CI, confidence interval.

RESULTS (Ki), estimated from these data by the method of Cheng and Prusoff,49 were in good agreement with previously published Human mAChR M4 affinity constant data for these ligands, either at human M4 receptor or in mammalian tissues (Table 1). Carbachol treatment of transiently transfected cells resulted in an increased induction of CRE-Luc–mediated luminescence Chicken mAChR M4 (maximum 87-fold increase) with an EC50 ¼ 5.6 lM; 10 lM was selected as the fixed agonist concentration, as it resulted in a Carbachol treatment resulted in an increased induction of CRE- submaximal induction of CRE-Luc (approximately 80% of Luc–mediated luminescence (maximum 287-fold increase), 44 maximum response). Relative IC50 values of mAChR antago- similar to results reported previously, with an EC50 ¼ 2 lM; nists, ranked in order from highest to lowest potency at human 10 lM was chosen as the fixed concentration, as it gave a large mAChR M4, were QNB (115 pM) > atropine (390 pM) ‡ but submaximal activation of CRE-Luc induction (approximate- oxyphenonium (400 pM) > mepenzolate (1 nM) > MT3 (8 nM) ly 90% of maximum response). The behavior of antagonists at > himbacine (10 nM) > dicyclomine (15 nM) > pirenzepine the cM4 receptor did not mimic perfectly their behavior at (23 nM) > tropicamide (155 nM) (Fig. 1). Inhibitory constants human M4. Relative IC50 values for cM4, in order from highest

FIGURE 2. Inhibitory potencies of various mAChR antagonists at the chicken cM4 receptor. Antagonists are categorized into full myopia inhibition (open/colored symbols), partial myopia inhibition (partially filled symbols), and no myopia inhibition (filled symbols) according to previously 14,16,19 published research, and ranked in descending order from most to least potent in our receptor assay (IC50 6 95% CI). MT3 (yellow) and atropine (blue) are indicated separately as they are the most researched anti-myopia mAChR antagonist in humans (atropine), and the most potent anti-myopia drug found to date in chicks (MT3). ATR, atropine sulfate; OXY, oxyphenonium bromide; PRZ, pirenzepine dihydrochloride; CI, confidence interval.

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FIGURE 3. Inhibitory potencies of mAChR antagonists at hADRA2A. Antagonists are categorized into full myopia inhibition (open/colored symbols), partial myopia inhibition (partially filled symbols), and no myopia inhibition (filled symbols) according to previously published research,14,16,19 and ranked in descending order from most to least potent in our receptor assay (IC50 6 95% CI). MT3 (yellow) and atropine (blue) are indicated separately as they are the most researched anti-myopia mAChR antagonist in humans (atropine), and the most potent anti-myopia drug found to date in chicks (MT3). In this assay, dicyclomine is indicated by an open circle with a cross, because it had no activity at the hADRA2A receptor at the concentrations tested. YOH, yohimbine hydrochloride; ATR, atropine sulfate; OXY, oxyphenonium bromide; PRZ, pirenzepine dihydrochloride; CI, confidence interval.

to lowest potency, were QNB (310 pM) > oxyphenonium (560 affinities much lower than those with which they interact with pM) > atropine (710 pM) > mepenzolate (4 nM) > himbacine mAChRs. The noteworthy exception was MT3, which bound (6 nM) > dicyclomine (41 nM) > pirenzepine (49 nM) > to hADRA2A with an inhibitory potency (IC50 ¼ 15 nM) tropicamide (245 nM) > MT3 (450 nM) (Fig. 2). When comparable to that for its interaction with the human M4 converted by the Cheng-Prusoff correction, the IC50 data for mAChR (IC50 ¼ 8 nM). atropine, QNB, and pirenzepine are in good agreement with previously published Ki values obtained by utilizing the same Controls receptor clone38,51 (Table 1). Although most antagonists acted Three different control experiments were conducted to verify with comparable relative inhibitory potencies at the cM4 that the changes in CRE-Luc, reflecting increases in intracel- receptor and the human M4 receptor, the Ki for MT3 in lular cAMP, were due to activation of the transfected receptor blocking cM4 was 56 times less potent (IC50 ¼ 450 vs. 8 nM, respectively; P < 0.0001, unpaired t-test, 2-tailed). Thus, and not stimulation of endogenous receptor signaling path- relative to all other mAChR antagonists tested, MT3 was the ways. First, cells were transfected with the pcDNA 3.1(þ) plasmid only. There was no significant change in luminescence weakest antagonist at chick cM4. in response to carbachol or clonidine treatment (Supplemen- tary Fig. S1). Second, the impacts of elevating intracellular Human a2A-Adrenoceptor calcium using a calcium ionophore (CI A23187), or activating Clonidine treatment of cells transiently transfected with protein kinase C with phorbol 12-myristate 13-acetate (PMA) hADRA2A resulted in increased CRE-Luc–mediated lumines- were tested. CI A23187 treatment, resulting in an increase in intracellular Ca2þ, could in principle cause activation of Ca2þ- cence (maximum 26-fold increase), with an EC50 ¼ 430 nM; 1 lM was chosen as the fixed concentration, as it gave a dependent cyclase and an increase in cellular cAMP, thereby submaximal activation of CRE-Luc (approximately 78% of mimicking the action of Gq-coupled GPCRs (Supplementary maximum response). Although increased cAMP synthesis is not Fig. S2). Alternatively, the protein kinase C (PKC) activator, the usual response to agonist treatment of G -coupled PMA, could mimic the potential action of Gq-protein–coupled i receptors via phospholipase action, and raise both inositol receptors, this Gs-coupling effect has been reported previously in CHO cells transfected with the hADRA2A receptor (also triphosphate and diacylglycerol levels by activating cAMP 43 response element binding protein (CREB)—thus causing known as a2C10). Relative IC50 values for the mAChR antagonists at the hADRA2A receptor, ranked in order from increased luminescence without an increase in cellular cAMP highest to lowest potency, were MT3 (15 nM) > himbacine (17 (Supplementary Fig. S2). Importantly, we observed no signif- icant change in Luc with treatment of either CI A23187 (1 lM) > atropine (45 lM) > oxyphenonium (463 lM) > N lM) or PMA (10 lM), especially when compared to the pirenzepine (867 lM) > tropicamide (1.5 mM) > QNB (260 response resulting from agonist treatment of transfected cells lM; maximum 82% inhibition) > mepenzolate (798 lM; (Supplementary Fig. S2). maximum 68% inhibition) >>dicyclomine (no detectable activity) (Fig. 3). The IC50 for yohimbine, an a2-adrenergic antagonist, was 5 nM (calculated K ¼ 2 nM). These data are i(CP) DISCUSSION in good agreement with previously published values for the 29,52 affinity of MT3 (Ki(CP) ¼ 5 nM) and yohimbine (Ki ¼ 400 The primary finding of our study is that the relative potencies pM–6 nM)53–55 at the hADRA2A receptor (Table 2). Thus, our of myopia-inhibiting mAChR antagonists to block the adrener- data demonstrated unequivocally that myopia-inhibiting gic hADRA2A receptor are correlated better with their ability mAChR antagonists can bind to hADRA2A, although with to inhibit chick FDM than are the relative inhibitory potencies

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TABLE 2. Binding Data for a2-Adrenergic Drugs and mAChR Antagonists at hADRA2A

Drug Ki ED50 Study Ki(CP) Study Ki /ED50 Literature Inhibits Myopia Est. [Vit] Source Cat No.

Alpha2-adrenergic drugs at hADRA2A YOH 5 nM 1 nM *400 pM–6 nM53–55 No, 0.01–1 mM Sigma Y3125 †630 pM128 Clon 425 nM N/A *60–160 nM129 Yes, 0.01–1 mM Sigma C7897 †4 nM130

Drug IC50 Study Ki(CP) Study Ki Literature Inhibits Myopia Est. [Vit] Source Cat No. Muscarinic antagonists at hADRA2A ATR 45 lM14lM 1–100 lM30,31 Yes16, 0.1–10 mM Sigma A0257 MT3 15 nM 5 nM 3 nM29,52 Yes14,15, 10–350 nM Peptides International PMT-4410-s HIM 17 lM5lM N/A Yes19, 3–12 mM Enzo Life Sciences ALX-550-061 OXY 470 lM 140 lM N/A Yes16, 1–10 mM Sigma O5501 PRZ 870 lM 250 lM N/A Yes16, 10 mM Sigma P7412 QNB 260 lM (82%) 80 lM N/A Partial16, 0.74 mM Sustainable Scientific BR-143013 TRP 1.5 mM 440 lM N/A Partial16, 10 mM Sigma T9778 MEP 780 lM (68%) 240 lM N/A No16, 1–10 mM Sigma M5651 DIC No binding N/A N/A No16, 1–10 mM Sigma D7909

128 130 31 Non-ADRA2A-specific receptors/tissues: chick pineal, chick cortex, rabbit pulmonary artery. Ki/ED50 Study and IC50 Study data are the 49 data determined by our binding assay, Ki(CP) data are calculated from the IC50 values in our assay by the method of Cheng and Prusoff, and Ki/ED50 Literature and Ki Literature data are data from previously published studies. Maximum inhibition achieved was 100%, unless indicated otherwise by percent values in parentheses beside the reported IC50. YOH, yohimbine hydrochloride; Clon, clonidine hydrochloride; ATR, atropine sulfate; OXY, oxyphenonium bromide; PRZ, pirenzepine dihydrochloride; Est. [Vit], estimated vitreal concentrations, assuming uniform dilution in total vitreal volume, required for myopia inhibition in the chick; N/A, not available. * Human or mammalian mAChR M4 receptor or tissue. † Avian mAChR M4 receptor or tissue.

59 of the same compounds to block the muscarinic M4/cM4 pirenzepine. At 1 hour after injection, reported concentra- receptors (Tables 1, 2; Fig. 4). The data correlating the relative tions (percentage of the original injected amount) were 6% in abilities of the mAChR antagonists for myopia inhibition versus the retina, 1.5% in the choroid, and 2.5% in the sclera.59 Based their relative Ki values for blocking the chick and human on these data, and considering that 100 mM was used to mAChRs show MT3 as an obvious outlier (Figs. 4a, 4b, yellow achieve full myopia inhibition in the chick,16 estimated circles). Yet, the same correlation plot paradigm shows that the concentrations for ocular tissues would be retina ¼ 6 mM, relative Ki of MT3 to block hADRA2A is tightly correlated, as choroid ¼ 1.5 mM, and sclera ¼ 2.5 mM. These estimated are the Ki values of the other mAChR antagonists, with the concentrations greatly exceed those that would be expected to ability to attenuate chick FDM (Fig. 4c, yellow circle). These have mAChR-only effects, and fall within the range of findings support the hypothesis that myopia inhibition by high- concentrations that would enable pirenzepine to block the concentration mAChR antagonists may be mediated by hADRA2A receptor under in vitro conditions, as calculated by our assay (K ¼ 250 lM; Table 2). receptors other than mAChR M4/cM4, and our data point to i(CP) members of the a-adrenoceptor family as possible alternatives. In humans, atropine for myopia inhibition is delivered in the form of daily eye drops (0.01%–1%). Under experimental A secondary finding is that, although the relative inhibitory 3 potencies of most myopia-inhibiting mAChR antagonists for conditions, delivery of a single dose of 2% [ H]-atropine to the blocking the chick cM compared with the human M conjunctival sac of albino rabbits resulted in a distribution of 4 4 the original concentration of 0.09% in the sclera, 0.05% in the receptors are in good agreement, MT3 is an anomaly; it has 60 markedly reduced K for the chick cM receptor compared choroid, and 0.008% in the retina, 1 hour after instillation ; i 4 similar results (retina: 0.01%; choroid and sclera 0.38%) were with the human M receptor. This result is likely due to 4 seen 30 minutes after topical application of [C ]-atropine.61 differences in the ligand-binding sites on the two receptors. 14 Therefore, the concentrations of atropine in the eye after a single topical application of 0.01% to 1% (0.13–13 lM in the Distribution of Drugs Within Ocular Tissues After sclera, 0.07–7 lM in the choroid, and 0.01–1.1 lM in the Different Delivery Methods retina, extrapolated from single-dose studies in albino rab- bit60,61) are within the range of what we have found to be the A question to ask about the use of atropine for myopia IC50 for atropine’s ability to block the hADRA2A in in vitro cell inhibition (in humans and animals) is, Does the high culture preparations (our value ¼ 45 lM; value reported by concentration of antagonist, as administered, reflect its others ¼ 1–10 lM).30,31 The ocular distribution of atropine concentration at the site of action in the eye? Currently, the after multiple topical doses has not been determined, but site of action for atropine-mediated myopia inhibition is studies monitoring the accumulation of atropine in humans unknown, but best guesses identify the retina, choroid, and/ have shown that atropine buildup can occur. Serum levels of 56 or sclera. In chicks, intravitreal injection is the usual method atropine were undetectable after a single inhalation dose of 12 of delivery, and concentrations of 1 to 100 mM have been used to 25 ng/lL atropine sulfate, but were raised to 1.13 to 5.23 for mAChR antagonist-mediated inhibition of FDM16,57,58 (Table ng/lL when treatment was administered every 4 to 6 hours 1). Unfortunately, no studies exist on the ocular distribution of over a period of 48 hours.62 Pigmented tissues (RPE, iris, ciliary atropine after intravitreal injection into chick eyes; but one body, choroid, and retina) are especially apt at retaining study has investigated the ocular distribution, in chicks, of a atropine, which may lead to an initially weaker but significantly single dose of intravitreally injected radioactively labeled prolonged effect.63,64 It is possible, then, that topical

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FIGURE 4. Correlations between rank order inhibitory potency (x-axis) and rank order inhibition of axial elongation in chicks with experimentally induced FDM (y-axis) for mAChR antagonists at M4 (a), cM4 (b), and hADRA2A (c). Rank orders for both parameters are 1 ¼ least potent (pIC50)or effective (% axial length inhibition) and 9 ¼ most potent (pIC50) or effective (% axial length inhibition). A line of unity (slope ¼ 1) has been superimposed upon the graphs for visual reference. Inhibitory potencies of mAChR antagonists, and their abilities to inhibit FDM in the chick, correlate significantly for hADRA2A only (r ¼ 0.87, P < 0.01; Spearman’s ranked correlation). Atropine and MT3 are shown in color (atropine ¼ blue, MT3 ¼ yellow). Full inhibition of axial elongation (open/colored symbols), partial inhibition of axial elongation (partially filled symbols), and inhibition of axial elongation (filled symbols) according to previously published research in the chick.14,16,19

application of 0.01% atropine daily for long periods (up to 2 Difference in Binding of MT3 at M4 Versus cM4 years) could result in ocular concentrations much higher than those seen after a single dose, with intracellular organelles When attempting to assign functional consequences to such as melanin granules acting as reservoirs for slow release of treatment with a particular ligand, it is important to consider the drug and possibly providing local concentrations that the mechanism of binding of that ligand to its target receptor. Generally, orthosteric binding sites, where the transmitters or would be sufficient to interact with non-mAChRs. primary signaling molecules bind, are highly conserved (100%) Pirenzepine has also been tested for the treatment of between receptors of the same class and between species.71 childhood myopia, with twice-daily applications of concentra- ‘‘Selective’’ modulatory ligands such as MT3, however, will tions up to 2%; but the results did not support superiority of bind to receptor amino acid residues that are less conserved, clinical efficacy of pirenzepine treatment over atropine 65–68 usually outside the orthosteric pocket, in the extracellular treatment, and further studies have seemingly been loops (EL2 and EL3) of the target GPCR.39 In such instances, abandoned.69 Ocular distribution of a single dose of topically 3 substitution of even a single amino acid can have a significant applied [ H]-pirenzepine (2%) has also been tested in the rabbit impact on ligand–receptor binding characteristics.39,40 Thus, eye; concentrations in the ocular tissues 1 hour after while decreased conservation of receptor sequences in the administration were roughly half those calculated for atropine extracellular loops aids in selectivity of a ligand for a certain (retina: 0.004%, choroid: 0.021%, sclera: 0.052%)—possibly receptor subtype, the increasingly stringent requirements for because atropine, being more lipophilic, is better at penetrat- binding can also result in loss of activity at the same receptor ing the ocular tissues and is lost less rapidly than pirenze- subtype in other species. This finding is what we observed in 60 pine. The estimated concentrations of pirenzepine delivered our assay: MT3 bound poorly to cM4 compared to M4. Chicken to ocular tissues should be sufficient to activate all mammalian and human M4 share only a 71.4% to 72.7% sequence identity mAChR subtypes fully,70 but are expected to fall below the in EL3 and EL2, respectively (Supplementary Fig. S3). concentration required to achieve a significant blockade of Therefore, it is likely that the decreased activity of MT3 at hADRA2A signaling (retina: 2 lM, choroid: 12 lM, sclera: 30 cM4 is caused by amino acid differences in its EL2 and EL3 lM) as calculated by our assay (Ki(cp) ¼ 250 lM, Table 2). sequences, compared to those of M4. All other drugs tested in our assay did not differ significantly in their binding behavior at mAChR Antagonists at M ,cM, and hADRA2A M4 versus cM4. This result was expected; these ligands are 4 4 known to be orthosteric inhibitors of ACh at mAChRs, and the Receptors: Correlations With Myopia Inhibition reported orthosteric binding site of the mAChR M4/cM4 71 The relative inhibitory potencies of myopia-inhibiting mAChR receptors is 100% conserved between human and chick (Supplementary Fig. S3). antagonists at the hADRA2A receptor, but not at the chick cM4 and human M4 mAChRs, are well correlated with their reported ability to inhibit chick FDM14,16,19 (Fig. 4). We have Ocular a-Adrenoceptors and Possible Mechanism confirmed previous reports of high-affinity binding of MT329,52 of Action (Ki(CP) ¼ 5 nM) to hADRA2A, and demonstrated that atropine and himbacine can also bind to this receptor, albeit with Sympathetic innervation in the vertebrate eye services potencies much lower than their relative K values for blocking structures such as the dilator pupillae, the ciliary muscle and i epithelium, and ocular blood vessels within and outside the the mAChRs. All other mAChR antagonists tested had very low 72 choroid. Alpha2-adrenoceptors are best known as autorecep- potency for blocking hADRA2A, but 100% inhibition of 41,73 clonidine-mediated effects was achieved with oxyphenonium, tors, but they can also act as heteroreceptors. Alpha2- adrenoceptors that act as autoreceptors are located on tropicamide, and pirenzepine—drugs that were reported to presynaptic adrenergic neurons; they are activated by epi- inhibit myopia fully in the chick when used at high nephrine/norepinephrine and inhibit the release of their own concentrations. The maximum inhibitory effects achieved by neurotransmitters. Alpha2-adrenoceptors that act as hetero- QNB (partial myopia inhibition in chick) and mepenzolate (no receptors are located presynaptically on nonadrenergic neu- myopia inhibition in chick) were 82% and 68%, respectively, rons, and there is evidence that a2-adrenoceptors can modulate and dicyclomine (no myopia inhibition in chick) had no the release of neurotransmitters such as GABA, dopamine, and discernable effect at the concentrations tested. At the human serotonin in the rat brain and retina.74–79 Interestingly, both and chick M4/cM4 receptors, atropine, QNB, mepenzolate, and GABA and dopamine have been implicated in the regulation of dicyclomine are all comparably potent inhibitors, with Ki eye growth.80–88 In the rat retina, the epinephrine-synthesizing values in the nanomolar range; yet atropine is the only one of enzyme phenylethanolamine N-methyltransferase (PNMT) has 16 these that can inhibit chick FDM. Most interesting is the been localized to amacrine cell populations89 and retinal behavior of MT3 at M4/cM4. It is the most effective drug found epinephrine levels have been reported to rise upon light 14,15 to date for mitigating chick FDM, and we confirmed its exposure90,91; but there is no further evidence for a role of high potency for the human M4 receptor. However, we found it epinephrine or norepinephrine in retinal visual processing of to be the least potent of all drugs tested at the chick cM4 any vertebrate species examined. In spite of this, a-adreno- receptor. This disconnect, between the potency of MT3 at cM4 ceptors are present on retinal neurons. In rat retina, human, and its effectiveness against chick FDM, supports our and primate tissue, ADRA2A immunoreactivity has been hypothesis that myopia inhibition is not mediated by the M4/ localized to the nonpigmented ciliary epithelium, cornea and 92 cM4 receptor. conjunctiva, and retinal ganglion cells and amacrine cells. In

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a transgenic mouse expressing an N-terminal hemagglutinin atropine, and have been associated strongly with reduction of 57 epitope (HA)-tagged a2A-receptor, driven by the endogenous eye growth. ADRA2A gene locus, ADRA2A gene expression was localized to horizontal cells, inner nuclear layer, inner plexiform layer, and Caveats and Considerations the retinal ganglion cell layer (Rao SS, et al. IOVS 2011;52:AR- VO E-Abstract 2059). In chick, immunoreactive ADRA2A has There are some discrepancies in the ability of mamba toxins been localized to retinal Muller¨ cells, amacrine cells, and to inhibit myopia in different animal models. Although MT3 ganglion cells (Costa GV, et al. IOVS 2012;53:ARVO E-Abstract inhibits FDM effectively in chicks14 and tree shrews,17 it 6545).93 seems to be ineffective at inhibiting the axial elongation Ocular a2-adrenoceptors are implicated in the pathogenesis induced by lens-induced myopia (LIM) in the same ani- 15,17 of glaucoma, and a2-adrenoceptor agonists, such as brimoni- mals. Conversely, MT7, a mamba toxin subtype that is dine and apraclonidine, are clinically approved for manage- highly selective for mAChR M1 and is not known to act at any ment of intraocular pressure (IOP). The prevalence of other receptor,28,29 inhibits LIM, but not the axial elongation glaucoma is said to be correlated with the prevalence of induced by FDM, in the tree shrew.17 MT7 has no effect in the 94,95 119 myopia, and new evidence suggests that these a2- chick, which is expected because chicks do not possess adrenoceptor agonists can inhibit myopia in chicks (Carr BJ, the M1 subtype. Pirenzepine, however, is effective at Stell WK. IOVS 2016;57:ARVO E-Abstract 4738) and guinea inhibiting chick FDM, possibly due to binding to the cM 43 2 pigs. It is still unknown which neural circuits or receptors receptor, which MT7 does not recognize, or to off-target might mediate these effects, but Liu et al.43 suggested that binding at a2-adrenoceptors as evidenced by our work. These attenuation of IOP is responsible for inhibition of myopia in differences in treatment effectiveness of mamba toxins are in guinea pigs. A direct mechanical influence of IOP (within the line with evidence suggesting that the circuits involved in LIM physiological range) on axial elongation, as hypothesized by and FDM are in some ways different,120–122 and that— Pruett96 and others,97–99 does not seem likely, however. 100–103 whatever the mechanism affected by atropine (and other Human clinical studies have found no relationship, an myopia-inhibiting mAChR antagonists) is—the results of our increase in IOP after but not preceding the onset of 99,104 study may be unique to the circuitry of FDM, and not LIM. myopia, or a small decrease in IOP before onset of Although we have shown that some mAChR antagonists will childhood myopia103; animal studies have produced equally 97,105 bind hADRA2A at high concentrations—comparable to those equivocal results. A possible reason for this could be found to be effective against chick FDM—we have not ruled measurement bias induced by utilizing indentation or appla- out other a-adrenoceptor subtypes, non-M muscarinic sub- nation tonometry, which calculates IOP from the amount of 4 types (i.e., mAChR M1/cM2), or non-mAChR/nonadrenergic pressure required to flatten an area of the cornea. The receptor targets. The high concentrations of these drugs accuracy of tonometry is dependent on factors such as corneal required to inhibit FDM in the chick model impede our ability thickness106 and scleral compliance,107,108 and increased to identify any single receptor as the key target for the impact scleral compliance (scleral creep) has been observed in of any compound on myopia, because of the distinct possibility humans with pathologic myopia109 and mammals with of many unidentified off-target receptor-mediated effects.21 experimentally induced myopia.110,111 Thus, if the sclera is Furthermore, because of technical issues we could not more compliant than normal, it may lead to a mistaken overcome, we were unable to express a functional chick measurement bias toward lower IOP. In contrast, a2-adreno- ceptor agonists also were reported to effectively inhibit FDM in ADRA2A receptor in our HEK cell expression system, which chicks (Carr BJ, Stell WK. IOVS 2016;57:ARVO E-Abstract would have enabled us to compare the ‘‘nonspecific’’ affinities 4738), in which there is no increase in scleral compliance in of mAChR antagonists directly for the chick versus human a2A- response to myopiagenic stimuli111; IOP measurements were adrenoceptors. What can be concluded with confidence from not recorded in that study, however, so we cannot exclude a our data, however, is that M4/cM4 cannot be seen as the unique role completely. The relationship between the actions and or even the leading receptor candidate for mediating the receptor targets of the compounds we studied and their impact inhibition of FDM by atropine (and other mAChR antagonists) on IOP and myopia remains an interesting topic for future in the chick bioassay. work. Notwithstanding the possible role of IOP in myopia, our work establishes clearly that action of atropine via the mAChR Summary M receptor is highly unlikely to be a factor. 4 Our data provide new information about the relative potencies Alpha2-adrenoceptor agonism in the retina can also result in the upregulation of neuroprotective agents and mechanisms, of muscarinic antagonists at the human and chick mAChR M4/ such as an increase in basic fibroblast growth factor (bFGF) in cM4 receptors and the human a2A-adrenoceptor. This is the rat photoreceptors112,113 or activation of the extracellular first study to characterize the inhibitory action of oxypheno- signal-regulated kinase signaling pathway in chick Muller¨ nium, mepenzolate, MT3, dicyclomine, and tropicamide 93 cells. Interestingly, both bFGF and extracellular signal- explicitly at chicken cM4, and the first to test specifically and regulated kinase signaling have been implicated in protection report in detail, the potential ‘‘off-target’’ actions of multiple against chick FDM114,115 and guinea pig lens-induced myo- mAChR antagonists at hADRA2A. Consequently, these data pia.42,116,117 Finally, brimonidine might be involved in the provide a concentration range over which these drugs should regulation of nitric oxide synthase in retinal arterioles, where it be expected to act in a mAChR-specific manner, and a may activate endothelial nitric oxide synthase (eNOS), concentration range at which they can be predicted to have resulting in vasodilation, as reported in pigs.118 It is important ‘‘off-target’’ effects at hADRA2A and possibly other receptors. to note that although there are no intraretinal blood vessels in The focus of thinking about the anti-myopia action of atropine the chick retina, it is served by blood vessels in the choroid and as muscarinic, combined with the lack of knowledge of the an intravitreal structure called the pecten. Whether a2- mechanism of atropine’s therapeutic actions (in the setting of adrenoceptor ligands can also modulate NOS (neuronal NOS, either human or chick myopia), is a deterrent to the discovery eNOS, or inducible NOS) activity or expression in retinal of more effective anti-myopia agents with fewer side effects. neurons is worth investigating further, since synthesis and The data presented here make a compelling case for continued release of nitric oxide are necessary for myopia inhibition by investigation into non-mAChR targets for atropine-mediated

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myopia inhibition, with a-adrenoceptors being provocative 16. Luft WA, Ming Y, Stell WK. Variable effects of previously candidates for novel therapeutic interventions. untested muscarinic receptor antagonists on experimental myopia. Invest Ophthalmol Vis Sci. 2003;44:1330–1338. Acknowledgments 17. Arumugam B, McBrien NA. Muscarinic antagonist control of myopia: evidence for M4 and M1 receptor-based Supported by a Natural Sciences and Engineering Research pathways in the inhibition of experimentally-induced axial Council of Canada (NSERC) Discovery Grant (RGPIN/131-2013) myopia in the tree shrew. Invest Ophthalmol Vis Sci.2012; and the Foundation Fighting Blindness Canada EYEGEYE Research 53:5827–5837. Training Fund (WKS), a Canadian Institutes of Health Research 18. McBrien NA, Stell WK, Carr B. How does atropine exert its (CIHR) grant (MOP13759) (MDH), and an NSERC PGS-D award anti-myopia effects? Ophthalmic Physiol Opt. 2013;33:373– (PGSD2-476058-2015), Dr. D. Grant Gall Graduate Traineeship, 378. Queen Elizabeth II Scholarships, and Odd Fellow Rebekah Visual Research Fund Awards (BJC). Core support was provided by the 19. Cottriall CL, Truong HT, McBrien NA. Inhibition of myopia University of Calgary Core DNA Services and the Live Cell Imaging development in chicks using himbacine: a role for M4 Facility, funded by the Snyder Institute for Chronic Disease, at the receptors? Neuroreport. 2001;12:2453–2456. Cumming School of Medicine, University of Calgary. 20. Fischer AJ, Miethke P, Morgan IG, Stell WK. Cholinergic Disclosure: B.J. Carr, None; K. Mihara, None; R. Ramachan- amacrine cells are not required for the progression and dran, None; M. Saifeddine, None; N.M. Nathanson, None; W.K. atropine-mediated suppression of form-deprivation myopia. Stell, None; M.D. Hollenberg, None Brain Res. 1998;794:48–60. 21. Schwahn HN, Kaymak H, Schaeffel F. 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