Previous studies on both humans and animals have employed high doses of either the

broadband antagonist atropine or the partially selective M1/M4 muscarinic antagonist,

pirenzepine, in order to inhibit the development of myopia. 3–5,7,9,12 Previous studies,
including our own laboratory, have employed doses of atropine as much as 10,000 times
or higher than theoretically required for binding at the M4 receptors or 17,500 times or
higher than required for binding at the M1 receptor 5,43,44 based on published
receptor affinity values. 45 Consequently, a number of studies have questioned whether
muscarinic antagonist drug effects on myopia inhibition are working via retinal receptors
and proposed either a nonreceptoral mechanism of action or at muscarinic receptors in
the sclera. 16,46,47 The present study utilized highly selective muscarinic antagonists
(MT3 and MT7) in an animal model that has both M4 and M1 receptors, and used doses
to provide physiologically relevant concentrations at the receptor binding site for ocular-
based receptors, specifically retinal-based receptors. The finding that these highly
selective antagonists inhibited experimentally-induced axial myopia gives much greater
confidence in the hypothesis that muscarinic antagonist inhibition of myopia is initiated
via a receptoral-based mechanism. It is well established that MT3 and MT7 are highly
selective allosteric antagonists at the M4 and M1 muscarinic receptors, respectively.
26,48,49 These antagonists bind to the allosteric binding site and bring a change in the
affinity of the receptor for the endogenous orthosteric ligand (in this case acetylcholine).
The use of physiological concentrations is critical for appropriate conformation changes
at the respective receptor. For example, alcuronium has a biphasic effect on binding of
3H-NMS to M2 receptors, where low concentrations (10 μM) have agonist effects and
the high concentrations (1 mM) are antagonistic. 50 In addition, low concentrations (1–
10 μM) of gallamine are shown to increase the dissociation of [3H] quinuclidinyl
benzilate at M1 and M2 receptors, while 1 mM gallamine slows it. 51 Although to our
knowledge there is no study to date that has reported the above biphasic effect for MT3
or MT7 at their receptor subtypes, as noted the use of high concentrations can lead to
different pharmacological drug receptor effects than when using physiological
concentrations. The in vitro affinity constants indicate that MT7 has its effects at the M1
receptor at doses of approximately 0.1 nM and for the M4 receptors at doses of
approximately 2 μM. The present study employed a calculated maximum concentration
of 220 nM of MT3 and MT7 at the retinal/vitreous interface. However, the
concentration at the retinal, choroidal, or scleral muscarinic receptors would be less
than 220 nM, due to passage through ocular tissue barriers. Thus, it is highly likely that
the inhibition of myopia, due to reduced vitreous chamber elongation, by MT3 and MT7
was caused by pharmacological actions at their respective receptor targets with little or
no cross reactivity with other muscarinic receptors.