Myopia-Inhibiting Concentrations of Muscarinic Receptor Antagonists Block Activation of Alpha2a-Adrenoceptors in Vitro

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 atropine, a Health Sciences Centre B101, 3330 muscarinic acetylcholine 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, mamba 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: himbacine; PRZ: pirenzepine; TRP: tropicamide; OXY: oxyphenonium; QNB: 3- Citation: Carr BJ, Mihara K, Rama- quinuclidinyl benzilate; DIC: dicyclomine; MEP: mepenzolate) 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, muscarinic antagonist, 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 anticholinergic 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 acetylcholine receptor (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 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded from tvst.arvojournals.org on 10/04/2019 Off-Target Binding of Muscarinic Antagonists IOVS j June 2018 j Vol. 59 j No. 7 j 2779 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 choline 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

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