Impulses are transmitted across the spaces separating these structures by acetylcholine. A 45-kd glycoprotein, called the cholinergic neuronal differentiation factor, seems to play a role in determining which embryonic neurons will develop acetylcholine synthesizing properties.^1,2

The earliest pharmacologic studies were of those tissues innervated by the parasympathetic nervous system. Dixon³ pointed out that the application of muscarine produced effects that were identical to those produced by the stimulation of the vagal nerve. Loewi⁴ demonstrated that vagal nerve stimulation released a substance that he subsequently identified as acetylcholine. Dale5 was the first to suggest that the action of acetylcholine was terminated by an enzyme, an esterase, that hydrolyzed the molecule.

Cholinergic systems consist of a number of subcellular components: choline acetyltransferase; acetylcholine; sodium-dependent, high-affinity choline uptake system; clear vesicles; acetylcholinesterase; and nicotinic and muscarinic receptors.

CHOLINE ACETYLTRANSFERASE

The gene for choline acetyltransferase is located on the long arm of human chromosome 10.⁶ Within its length it also contains the gene that encodes the protein responsible for transporting acetylcholine into the vesicle. Although these two genes overlap, they do not share similar protein sequences.⁷

Choline acetyltransferase synthesizes acetylcholine from choline and acetyl-coenzyme A. Choline acetyltransferase is found in the soluble cytoplasm and attached to the outer membrane of acetylcholine-containing vesicles. It is not found within these vesicles.^8,9 The soluble form predominates at more physiologic pH.¹⁰ The binding of choline acetyltransferase to vesicle membranes is reversible and presumably due to ionic attraction between positively charged choline acetyltransferase and negatively charged membrane. For maximum enzymatic synthesis of acetylcholine, higher concentrations of choline than of acetyl-coenzyme A are required. For example, in human brain, the Michaelis' constant (Km) for choline is 510 μmol/L and the Km for acetyl-coenzyme A is 11 μmol/L.¹¹ Imidazole-containing compounds, such as pilocarpine, stimulate in vitro enzymatic synthesis of acetylcholine.^12,13 The possibility of small amounts of nonenzymatic production of acetylcholine by imidazoles has also been raised.¹⁴

Because choline acetyltransferase accumulates proximal to a nerve ligature, it has been assumed that the enzyme is transported by axonal flow from the nerve cell body toward nerve terminals.¹⁵ However, there appears to be some distal-to-proximal flow of enzyme as well.¹⁶

ACETYLCHOLINE

Acetylcholine is found in both the cytoplasm and vesicles of cholinergic nerve endings. When high concentrations of cytoplasmic acetylcholine exist, choline kinase is stimulated. Choline kinase phosphorylates choline and diverts it away from acetylcholine synthesis and toward phospholipid synthesis.¹⁷ In this way, acetylcholine may regulate the availability of its own substrate. The acetylcholine-containing vesicles are 30 nm to 60 nm in diameter and are electron transparent (i.e., “agranular”). They contain high concentrations of acetylcholine (approximately 47,000 molecules per vesicle [520 mmol/L]) and adenosine triphosphate (ATP) (approximately 17,000 molecules [170 mmol/L]); guanosine triphosphate (GTP) is present in 20 mmol/L concentration.¹⁸

When the neuron is at rest, acetylcholine is released spontaneously from the cytoplasm by a non-calcium-dependent mechanism. The amounts of cytoplasmic acetylcholine leaked are inversely regulated by neuronal membrane ATPase activity (i.e., the greater the ATPase activity, the lower the amounts). Evoked release of acetylcholine occurs from vesicles and depends on the presence of extracellular calcium.^19–21

Recycling of a vesicle (i.e., the time from acetylcholine release to refilling and re-release) appears to take approximately 1 minute.²² This cycle can be divided into eight steps²³:

1. Docking (vesicle contacts internal surface of synaptic axon membrane)
   
2. Priming (conformational change allowing calcium to trigger exocytosis of acetylcholine)
   
3. Exocytosis (calcium produces release of acetylcholine into the synaptic space)
   
4. Endocytosis (vesicle closes)
   
5. Translocation (vesicle is moved internally)
   
6. Endosome fusion
   
7. Neurotransmitter uptake
   
8. Translocation (vesicle returns near synaptic axon membrane)
   

A number of proteins in the membrane of the synaptic vesicle have been identified (e.g., synaptophysin and synaptotagmin).^24,25 The acetylcholine transporting protein identified on chromosome 10 contains approximately 40% of the amino acid sequences contained by these two vesicular proteins and is believed to have 12 transmembrane spanning domains. Thirty or more low-molecular-weight GTP-binding proteins, called Rab proteins, are involved in directing the vesicle through the cycle. One cluster, called the synaptosecretosome, triggers acetylcholine exocytosis; the key protein in this group is called mediatophore. Other proteins link the vesicle to the synaptic membrane. Some of these have channel structures that could form the route taken by released acetylcholine on its way to the synaptic space. This raises the possibility that direct or full fusion of the vesicle with the synaptic axon membrane is not needed.^26,27 A specific inhibitor of acetylcholine uptake into cholinergic vesicles, vesamicol, has been identified. Botulinum toxins attack some of the proteins linking the synaptic vesicle to the axon membrane and prevent calcium from triggering functional acetylcholine release.^28–30

CHOLINE UPTAKE

Acetyl-coenzyme A is synthesized in neuronal mitochondria from pyruvate, by pyruvate dehydrogenase, and transferred into the cytoplasm for acetylcholine synthesis. Choline is either synthesized by the neuron or obtained from the circulation.³¹ Neurons can synthesize choline by methylation of phosphatidylethanolamine or ethanolamine.^32,33 Phospholipase D hydrolyzes phosphatidylethanolamine to phosphatidic acid and choline.^34,35 Choline can be taken up by a neuron over its entire surface if a high concentration (i.e., Km = 10 to 100 μmol/L) is present.³⁶ This system is referred to as the low-affinity choline uptake system and probably plays little, if any, physiologic role. However, a sodium-dependent, high-affinity choline uptake system (i.e., Km = 1 to 10 μmol/L) is located on the synaptic nerve endings.³⁷ The high-affinity system becomes more active when the neuron is releasing acetylcholine and/or when it is synthesizing more acetylcholine.^38–40 Hemicholinium blocks the high-affinity choline uptake system. Although the choline transported into the nerve terminals is rapidly acetylated, there does not appear to be a direct linkage between the high-affinity system and choline acetyltransferase.⁴¹

RECEPTORS

There are two types of cholinergic receptors: muscarinic and nicotinic. Classically, these receptors were thought to exist only on the distal sides of the neuron-muscle fiber and neuron-neuron junctions (i.e., postjunctional receptors). A large amount of evidence indicates that they exist on the proximal sides of these junctions as well (i.e., the neuron terminal releasing acetylcholine has prejunctional receptors). Postjunctional receptors are effector receptors. Prejunctional receptors are involved in feedback stimulation and inhibition of transmitter release.

Postjunctional Receptors

NICOTINIC. Nicotinic receptors respond to acetylcholine and nicotine but not to muscarine. They are found in the central nervous system, in ganglia, and on striated muscle fibers. The specialized receptor area of a striated muscle fiber is termed the myoneural junction. This area is marked by infolding and convolutions of the muscle fiber membrane and a significant increase in the density of acetylcholinesterase molecules.

MUSCARINIC. Muscarinic receptors respond to acetylcholine and muscarine but not to nicotine. These receptors are found in the central nervous system, in ganglia, and on smooth muscle fibers. The junction of a parasympathetic neuron with a smooth muscle fiber is not highly specialized. The two are separated by a relatively wide distance, ranging from 15 to 1900 nm. This has functional significance (i.e., the narrower the cleft, the greater the concentration of transmitter at both prejunctional and postjunctional receptors). Junctional clefts in the iris sphincter are approximately 15 nm. Muscarinic reception may occur at those sites on smooth muscle fiber membranes where electrondense structures, 5 to 6 nm thick, are found.⁴² Muscarinic receptors have a latency of 100 to 500 msec, in contrast to nicotinic receptors, which have a latency of 30 to 100 msec. However, it does not appear that this latency is simply a function of the distance between nerve ending and receptor.⁴³

Nicotinic Receptor Structure. Nicotinic receptors have a molecular weight of approximately 250 kd. Each receptor consists of five glycopeptide chains (two of which are identical): two alpha, one beta, one delta, and one epsilon.⁴⁴ Some species variations exist.⁴⁵ Human fetal receptors are found diffusely along the muscle fiber membrane and have a gamma subunit instead of an epsilon. When stimulated, fetal receptors provide a membrane depolarization that has a longer duration and fetal receptors are less readily desensitized by prolonged agonist exposure. Conversion to the adult form occurs upon innervation when the receptors become clustered at synapses.⁴⁶ Extraocular muscles are an exception in that some of their muscle fibers retain the fetal type of receptor.^47,48 The fetal receptor is not found in the human adult levator palpebrae superioris of the lid.⁴⁹ In rats, all of the myoneural junctions of the multiply innervated (en grappe) extraocular muscle fibers, and a limited number of the singly innervated (en plaque) extraocular muscle fibers, co-express both the adult and fetal forms of the receptor.⁵⁰

Each glycopeptide subunit of the receptor spans the entire length of the receptor, and the five subunits form a pentagon around a central ion channel, which also runs the entire 11-nm length of the receptor.⁵¹ The receptor protrudes approximately 5.5 nm on the extracellular side of the cell membrane and approximately 1.5 nm on the intracellular side. The ion channel opening is widest extracellularly and then narrows as it passes through the cell membrane.⁵²

The binding site for cholinergic drugs on striated muscle nicotinic receptors is, at least in part, on the extracellular portion of the alpha subunit in a region containing two adjacent cysteines.^53–55 In neuronal nicotinic receptors, the binding site may be on the beta subunit.⁵⁶ Because there are two alpha subunits, there are two binding sites for acetylcholine on nicotinic muscle receptors. Both binding sites must be bound to elicit a response.⁵⁷ One is a high-affinity site and the other is a low-affinity site; the differences in affinity are conferred by the gamma and delta subunits where they contact the alpha subunits. The gamma subunit confers high affinity and the delta subunit confers low affinity.⁵⁸

The carbonyl side of the acetylcholine molecule reacts with the nicotinic receptor, whereas the methyl side reacts with the muscarinic receptor.⁵⁹ The acetylcholine molecules bind in a cooperative manner (i.e., binding of one acetylcholine molecule increases the affinity of the receptor to bind the second molecule).

Activating the receptor opens the ion channel for approximately 1 msec and produces a membrane depolarization of approximately 0.2 μV.⁶⁰ The length of time the channel is open depends on the agonist (e.g., acetylcholine = 1 msec; carbachol = 0.33 msec; and succinylcholine = 0.23 msec). The nicotinic ion channel is almost equally permeable to Na⁺ and K⁺ ions but impermeable to Cl- ions. Larger monovalent cations pass through the channel more readily than do smaller ones.⁶¹ This is reversed for divalent cations (i.e., smaller ones pass through more readily). Differences in potencies between agonists are due to affinity for the receptor and efficacy for opening the channel. Carbachol has a channel-opening efficacy that is about two thirds that of acetylcholine. Nicotinic receptors outside the synaptic area stay open about three times as long as those in the synaptic area. The activated ion channels of singly innervated (i.e., en plaque or fast) muscle fibers have characteristics similar to those of multiply innervated (i.e., en grappe or slow) muscle fibers.⁶²

Competitive nicotinic antagonists interfere with acetylcholine's binding onto the alpha subunits. Noncompetitive antagonists may allow the ion channel to open, but then block it.⁶⁰ The action of a specific drug may be complex. For example, curare is classified as a nondepolarizing, noncompetitive nicotinic antagonist. It binds to the receptor at a site other than where acetylcholine binds (i.e., it is not competitive with acetylcholine at this point). Once bound, curare exerts its main action by competing with acetylcholine for the site on which the latter acts to open the ion channel.⁶³ Thus, there are competitive and noncompetitive aspects to curare's action. Furthermore, high concentrations of a competitive agonist, such as concentrations of acetylcholine above 300 μmol or carbachol above 1 mmol, can enter and transiently occlude the ion channel.⁶⁴ Decamethonium and succinyldicholine act primarily by blocking open channels. Such channel blockers cannot be antagonized by cholinesterase inhibitors because the latter result in prolonged and increased endogenous acetylcholine concentrations, which, in turn, stimulate the receptor to keep the ion channel open and the antagonist blocking it.⁶⁰

Muscarinic Receptor Structure. Whereas a nicotinic receptor consists of multiple subunits arranged to form an ion channel, a muscarinic receptor is a single glycoprotein that activates a G protein (i.e., a guanine nucleotide binding protein). Muscarinic receptors belong to a G protein-linked “superfamily” of receptors that includes α₁-, α₂-, and β-adrenergic receptors and serotonin receptors. All members of the family have an undulating ringlike structure with seven hydrophobic transmembrane spanning groups. These transmembrane groups are linked by alternating cytoplasmic and extracellular loops.^65,66 The possibility exists that some muscarinic receptors consist of two subunits.^67,68 Different muscarinic receptors have approximately 40% identical amino acid sequences. Within the superfamily, this falls to approximately 4%, with only 19 amino acids conserved among all known members.

Five different muscarinic receptor genes and their different receptor products have been identified. These are commonly designated as m₁, m₂, m₃, m₄, and m₅. Pharmacologic evidence for the functional existence of these receptors in tissues exists for only four of them, designated M₁, M₂, M₃, and M₄.⁶⁹

In man, the m₁ receptor consists of 460 amino acids; m₂ has 466 amino acids, m₃ has 590 amino acids, and m₄ has 471 amino acids.^70,71 The lack of highly specific drugs for each receptor subtype has hampered the identification of the corresponding functional tissue receptors. M₁ receptors have a high affinity for the antagonists pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and a low affinity for the antagonists methoctramine and himbacine. M₂ receptors have the reverse affinities. M₃ receptors have a high affinity for 4-DAMP but a low affinity for pirenzepine.⁷² Carbachol has a much greater affinity for M₂ than for M₁ or M₄ receptors.⁶⁵ The formation of an ionic bond between the amine group of the agonist or antagonist and the aspartic acid on the extracellular loop of the third transmembrane spanning group seems to be a feature of all muscarinic receptors.⁷³ However, there may be more than just one drug binding site.⁷⁴ The third cytoplasmic loop is the major determinant for G protein coupling selectivity.^75,76

Prejunctional Receptors

Since 1959, hypotheses have existed to explain why nicotine produces adrenergic effects when applied to sympathetic nerve endings.⁷⁷ Whereas nicotine causes an increase in norepinephrine release, muscarine and pilocarpine inhibit norepinephrine release. Pilocarpine's effect can be blocked by atropine. There is now a large body of evidence showing that receptors are also present on nerve endings. For example, there is markedly less binding of muscarinic drugs after sympathetic nerve endings have been destroyed by 6-OH dopamine.⁷⁸ These presynaptic receptors are believed to play a role in feedback control of receptor release. In this manner, release of acetylcholine by parasympathetic nerve endings could alter adrenergic activity. It appears that the muscarinic receptor is activated at lower acetylcholine concentrations than is the nicotinic, but when both are stimulated maximally, the effect of the nicotinic receptor predominates. Thus, when sympathetic nerve endings are bathed in low concentrations of acetylcholine, there is little or no norepinephrine release. The addition of atropine causes increased norepinephrine release. At higher acetylcholine concentrations, additional norepinephrine is released as the nicotinic receptors are stimulated. The muscarinic receptors are believed to be of greater physiologic importance. The existence of prejunctional receptors may explain why systemic administration of muscarinic drugs in vivo causes blood vessel dilatation even though blood vessels are innervated almost exclusively by adrenergic fibers.

Muscarinic presynaptic receptors have been found on acetylcholine-releasing neurons of striated muscle.⁷⁹ These may mediate a decrease in the release of neuronal acetylcholine by a feedback mechanism.⁸⁰

A variety of prejunctional receptors, besides nicotinic and muscarinic ones, exist on acetylcholine-releasing neurons. Parasympathetic neurons have prejunctional alpha receptors that, when stimulated, may inhibit acetylcholine release.⁸¹ Another prejunctional receptor appears to be sensitive to the purine nucleotide coenzyme A. Coenzyme A is a major product of acetylcholine synthesis. Coenzyme A has been reported to act as a presynaptic inhibitor of acetylcholine release and could modulate cholinergic activity.⁸² Prejunctional prostaglandin receptors may also exist on parasympathetic nerve endings. Prostaglandins are released during parasympathetic activity⁸³ and are reported to both facilitate and inhibit acetylcholine release.^84,85 However, there are also reports that prostaglandins have no effect on parasympathetic motor neurons.^86,87

INTRACELLULAR EFFECTS OF MUSCARINIC AGONISTS. Activated muscarinic receptors couple to specific intracellular proteins. This GTP-requiring coupling results in enzyme activation. Depending on the receptor subtype, phospholipase C may be activated and/or adenylcyclase activity increased or inhibited. Different effects have been reported by different laboratories.^71,73 A consensus evaluation is that m₁, m₃, and m₅ receptors strongly activate phospholipase C; m₂ and m₄ receptors strongly attenuate adenylcyclase activity while, to varying degrees, weakly increasing phospholipase C activity. Phospholipase C hydrolyzes phosphatidylinositol 4,5-biphosphate into myoinositol 1,4,5 triphosphate- and 1,2-diacylglycerol.⁸⁹ In muscle cells, the most important intracellular response ultimately produced may be release of intracellular stores of calcium resulting in cell membrane depolarization and, as a secondary event, entry of extracellular calcium.⁹⁰

Supersensitivity

In normally innervated striated muscle, nicotinic receptors are found almost exclusively at the motor endplate region (i.e., the density of receptors at myoneural junctions is more than 2500 times that at extrajunctional sites).⁹¹ Slow-twitch muscle fibers, such as the multiply innervated en grappe extraocular muscle fibers, tend to have more extrajunctional receptors than singly innervated fast-twitch fibers.^92,93 Extrajunctional receptors have a short half-life of approximately 19 hours, whereas those in the myoneural junction tend to be more stable with a half-life of approximately 12 days.⁹⁴

Denervation reduces the normal motor endplate nicotinic receptor half-life to approximately 3 days.⁹⁵ These receptors are subsequently replaced by receptors having a shorter half-life, similar to that of the extrajunctional receptors of innervated muscle.⁹⁶

When skeletal muscle is denervated, the number of receptors increases and they spread out from the myoneural junction.^97,98 Approximately 3 to 4 days after denervation, receptors appear outside the original synaptic area. Their number is maximum in approximately 2 weeks. After 6 to 8 weeks of denervation, extrajunctional receptors are no longer found in the degenerating fibers.⁹⁹ Electrical stimulation prevents this spread of receptors and reduces supersensitivity to acetylcholine.^100–102 Disuse can produce a small increase in the number of receptors even if innervation is present.^103,104

Denervation sensitivity in nicotinic structures may be associated not only with an increase in receptors but also with a decrease, or increase, in acetylcholinesterase activity.^105–107 A decrease would explain why sensitivity to acetylcholine increases by a factor of 1000 while the number of receptors increases only 20- to 30-fold.¹⁰⁸

The mechanism of parasympathetic muscle fiber supersensitivity is not as clear. There is some evidence that the number of receptors increases, although not dramatically.¹⁰⁹ However, binding studies are unable to distinguish between presynaptic and postsynaptic receptors. Thus, the number of postsynaptic receptors could increase and the number of presynaptic receptors could decrease with little overall change in total binding. There is evidence that a fall in acetylcholinesterase activity occurs.¹¹⁰ Additional causes of supersensitivity for which there is evidence are (1) the receptors formed after denervation may be qualitatively different¹¹¹ and (2) denervation may lead to a less stable muscle membrane, as shown by an increased sensitivity to extracellular ions such as potassium.¹¹²

Subsensitivity

Sustained activation of nicotinic or muscarinic receptors can result in subsensitivity (i.e., decreased muscle contraction on re-exposure to the agonist).¹¹³ In nicotinic receptors, desensitization has been associated with increased protein kinase A activity.¹¹⁴

In smooth muscle, prior atropine treatment prevents this effect. Pilocarpine stimulation does not reduce the number of receptors.¹¹⁵ Short-term (less than 1 hour) desensitization seems due to decreased agonist binding and/or inactivation of the intracellular coupling to guanylate cyclase. Longer-term desensitization seems due to decreased number and affinity of receptors. The half-life for receptor disappearance is 4 hours. The return of receptor sites (half-time = 6 hours) when acetylcholine is removed is more rapid than the return of their function (half-time = 16 hours).¹¹⁶

Muscarinic receptor desensitization is associated with two phenomena. One is phosphorylation of the receptor by a G protein-coupled kinase, which allows binding by a member of the arrestin family of proteins. Arrestin binding prevents coupling of the receptor with other G proteins. The major site for phosphorylation is on the receptor's third intracellular loop; there appear to be multiple phosphorylation sites on this loop.¹¹⁷ The other desensitization phenomenon is sequestration (i.e., internalization of the receptor away from the cell surface). Although sequestration may be facilitated by phosphorylation,¹¹⁸ the two phenomena occur by independent mechanisms.¹¹⁹

ACETYLCHOLINESTERASE

Acetylcholinesterase has been found in all cell membranes examined, even red blood cells.¹²⁰ Its function in cholinergic systems is to terminate the action of acetylcholine by hydrolyzing the transmitter to choline and acetic acid. Acetylcholinesterase can also hydrolyze the peptide bonds found in neuropeptides, such as substance P and enkephalins.¹²¹ In addition, the acetylcholinesterase-containing protein molecule may also contain other enzymatic functions, such as Na-K activated ATPase.¹²² It may, therefore, play a role in the membrane permeability changes that occur after acetylcholine-receptor interaction.¹²³

Acetylcholinesterase first binds the positively charged acetylcholine molecule at a negatively charged site, the “anionic” site. This anionic site is located in a cleft in the enzyme. The ionized rings of aromatic amino acids line the cleft and form the anionic site. Close to the anionic site is the catalytic “esteradic” site where hydrolysis occurs.¹²⁴ This esteradic site is also located in the cleft, approximately 2 nm deep (i.e., halfway through the full thickness of the enzyme). The esteradic site itself consists of a triad of amino acids: a serine, a histidine, and a glutamic acid.¹²⁵ After the anionic site has attracted, bound, and oriented acetylcholine, the esteradic site hydrolyzes acetylcholine in the sequence: acetylcholine-acetylcholinesterase → acetyl-acetylcholinesterase + choline → acetylcholinesterase + acetic acid + choline.

In addition, there is another anionic site on acetylcholinesterase located more distal from the esteradic site.¹²⁶ It may help guide acetylcholine to the active cleft in the esterase. Binding to this secondary anionic site by some drugs seems to inhibit the action of the esteradic site, which may account for why large concentrations of acetylcholine inhibit acetylcholinesterase. However, other drugs (e.g., atropine), upon binding to the secondary anionic site, appear to activate the esteradic site. Edrophonium (Tensilon), procaine, curare, tetracaine, and decamethonium only seem to bind the anionic site that is near the esteradic site.¹²⁷

Edrophonium, like acetylcholine, has a quaternary amine that interacts with an indole ring of a tryptophan molecule located at the primary esteradic site.^128,129 This places the meta hydroxyl group of edrophonium between the serine and histidine in the catalytic site, further blocking acetylcholine's approach.¹³⁰ The phosphorylating and carbamylating cholinesterase inhibitors form covalent bonds with the serine in the esteradic site.¹³¹ Demecarium has additional potency because it inhibits the esteradic site and because its two nitrogen atoms interact with both the primary and secondary anionic sites, which are 1.4 nm apart.¹³²

Acetylcholinesterase can be inhibited by a number of drugs. The result of this inhibition is that endplate potentials become more prolonged because individual acetylcholine molecules can repeat their effect.¹³³ Inactivation of acetylcholinesterase causes the half-decay times of the endplate currents to increase from 1.5 to 2 msec to 3 to 10 msec. Acetylcholinesterase inhibitors may also act by competing with acetylcholine for nonspecific binding sites, thereby increasing unbound levels of acetylcholine.¹³⁴

Acetylcholinesterase inhibition by organophosphorus compounds is irreversible unless the esteradic site is rapidly dephosphorylated by nucleophilic agents such as pyridine oximes.¹³⁵ If reactivation is not rapid, the phosphorus group can no longer be removed by nucleophilic agents. The amount of acetylcholinesterase that can be reactivated decreases exponentially with time because of a process called “aging.”¹³⁶ Aging occurs because of the release of an oxygen-bounded alkyl group from the enzyme-phosphorus complex.^137,138 The rate of aging depends on the organophosphorus compound used.¹³⁹ It can be retarded by prior administration of quaternary amines^140,141 and imidazole-containing compounds, of which pilocarpine is an example.^142,143 By using organophosphorus compounds, an estimate can be made of the rate of acetylcholinesterase synthesis (e.g., 50% of brain acetylcholinesterase activity has recovered 1.2 to 5.3 days after subcutaneous injection of soman).¹⁴⁴ Cholinesterase inhibitors differ in their abilities to penetrate the cell membrane. Those that can penetrate will inhibit cytoplasmic as well as cell membrane acetylcholinesterase. Diisopropyl fluorophosphate (DFP, isoflurophate) is uncharged and can penetrate the cell membrane well. The recovery of cholinesterase activity is therefore longer (7 to 11 days) after DFP administration.^145,146

Cholinesterase exists in two forms, asymmetric and globular.¹⁴⁷ The asymmetric form consists of 1 to 3 catalytic subunits attached to a collagen-like tail 500 nm in length. Each of these catalytic subunits is a tetramer made up of 4 sub-subunits. Thus, asymmetric cholinesterase may contain 4, 8, or 12 sub-subunits. Each sub-subunit has a mass of 80 to 90 kd. Sub-subunits are joined together in pairs by a single covalent disulfide bond.^148–150 Two of these pairs are held together by hydrophobic forces to create a tetramer. The collagen-like fibrous tail is a triple helix that anchors the enzyme to the cell membrane. At synaptic sites, the acetylcholinesterase is the 3-tetramer asymmetric form. Denervation of striated muscle results in a decrease of this form.¹⁵¹

The globular form consists of 1, 2, or 4 of the sub-subunits. Globular forms are attached to the cell membrane in various ways, one of which is by attachment to phosphatidylinositol. Human butyrylcholinesterase (also referred to as pseudo- or serum cholinesterase) is 95% globular tetramers; the remaining 5% monomers and dimers are believed to be degradation products.

The substrate specificities of acetyl- and butyrylcholinesterases are not due to their asymmetric or globular form. For example, small amounts of globular tetramers of acetylcholinesterase can be detected in serum. Similarly, although asymmetric cholinesterases are found only in peripheral nerve and muscle, both asymmetric acetylcholinesterase and asymmetric butyrylcholinesterase can be detected. Hybrids of acetylcholinesterase and butyrylcholinesterase exist. The tails of pure asymmetric acetylcholinesterase and pure asymmetric butyrylcholinesterase are significantly different in structure.¹⁵²

Smooth-muscle membranes contain the 3-tetramer asymmetric form of acetylcholinesterase as well as other forms. Although the absence of discrete myoneural junctions results in there being relatively less of the 3-tetramer asymmetric form compared with striated muscle fibers, it is interesting that the smooth muscles with the highest percent are the mammalian iris and ciliary body.¹⁵³

CHOLINE ACETYLTRANSFERASE

The corneal epithelium,¹⁵⁵ iris, ciliary body,¹⁵⁶ and retina¹⁵⁶ of some mammals, including humans, contain large amounts of choline acetyltransferase activity.¹⁵⁷ Nonmammals, such as turtles, have retinal photoreceptors containing choline acetyltransferase activity.¹⁵⁸ Ross and McDougal¹⁵⁹ have found that the inner plexiform layer contains the highest level of enzyme activity in mammalian retina.

ACETYLCHOLINE

Large quantities of acetylcholine have been detected in the corneal epithelium but not in the stroma or endothelium.^160–162 Investigations have attempted to link corneal acetylcholine with ion transport¹⁶³ and touch sensation.¹⁶⁴ Acetylcholine activity has also been detected in iris, ciliary body, retina, and choroid.^165–167 Dowling and Boycott,¹⁶⁸ using electron microscopy, have observed vesicles in retina that are similar to those associated with acetylcholine storage.

ACETYLCHOLINESTERASE

Significant levels of acetylcholinesterase have been found in the corneal epithelium, with lesser amounts in stroma.^169–171 Fresh primary aqueous and vitreous humors contain virtually no cholinesterase activity, but secondary aqueous or aqueous humor removed after eyes have been refrigerated 5 to 6 hours does contain increased activity, mainly that of butyrylcholinesterase.¹⁷² The increase in secondary aqueous activity is attributed to breakdown of the blood-aqueous humor barrier and to penetration of serum cholinesterase. The increase in cholinesterase activity on storage is attributed to postmortem cellular autolysis of tissues bordering the ocular fluids. Human sclera, iris, ciliary body, retina, choroid, and optic nerve also contain acetylcholinesterase.^173,174 Most of the iris enzyme activity is in the sphincter region.¹⁷⁵ In mammalian retinas, most acetylcholinesterase activity has been found in the amacrine cells of the inner plexiform layer.^176–178 Low concentrations of acetylcholinesterase, limited to the anterior capsular region, have been found in human lenses.¹⁷⁹ No cholinesterase activity has been found in retinal blood vessels.¹⁸⁰

RECEPTORS

Muscarinic receptors have been identified in rat extraorbital lacrimal gland.¹⁸¹ Their existence in corneal epithelium has been difficult to establish. They were not found in preparations of disrupted rabbit corneal epithelial cells^182,183 but were found in cultures of rabbit,^184–186 bovine,¹⁸⁷ and human¹⁸⁸ epithelium. Multiple subtypes of muscarinic receptors have been identified in rabbit corneal endothelium.^186,190 Messenger RNA for m₃ receptors has been found in human corneal epithelium, corneal endothelium, trabecular meshwork, anterior lens epithelium, iris epithelium, ciliary epithelium, and ciliary muscle.¹⁹¹ Muscarinic receptors are in mammalian iris sphincter and, to a lesser degree, iris dilator muscle.^192–195 These latter receptors may be part of a reciprocal cholinergic innervation.¹⁹⁶ Whereas mammalian iris contains muscarinic receptors, avian iris contains nicotinic receptors.¹⁹⁷

M₃ muscarinic receptors are present in rabbit,^198,199 bovine,²⁰⁰ and human iris sphincters.²⁰¹ M₁ receptors are present in ciliary processes.²⁰² M₃ receptors predominate in human ciliary muscle,²⁰¹ although the M₂ subtype is also present.¹⁹¹ In both the circular and longitudinal portions of the ciliary muscle there is strong expression of m₂, m₃, and m₅ messenger RNAs; the m₂ and m₃ messenger RNAs are more prominently expressed in the circular portion.²⁰³ M₃ receptors have also been identified in human trabecular meshwork cells.²⁰⁴

In mammalian retina, there is evidence that cholinergic receptors are present.^205–207 Muscarinic receptors are present in the human inner plexiform layer, and nicotinic binding sites have also been identified.²⁰⁸ In nonmammalian retina, nicotinic binding sites have been found in bipolar cell and amacrine cell synapses of the inner plexiform layer.²⁰⁹

CHOLINE UPTAKE

The corneal epithelium appears to have a low-affinity choline uptake system and not the high-affinity system associated with cholinergic nerve endings.²¹⁰ The retina, however, does have a high-affinity choline uptake system.^211–213 It is assumed that it is located in the membranes of the inner plexiform cells that synthesize acetylcholine. In addition, mammalian photoreceptor cells and lenses, which do not synthesize acetylcholine, also have a high-affinity choline uptake system.^214,215 Thus, cells that use large amounts of choline, as would be needed for phospholipid synthesis, may contain the high-affinity system.

CHOLINERGIC NEURONS

There are difficulties with each of the techniques used to determine which ocular neurons are cholinergic.

Denervation Studies

One method is to remove the cholinergic innervation to ocular structures and examine histologically for neuronal loss. Unfortunately, this begs the question because the experimenter first defines, a priori, which nerve trunks are cholinergic. For example, the cornea has been examined for parasympathetic innervation by removal of the ciliary ganglion.²¹⁶ However, a variable number of trigeminal nerve sensory fibers pass through the ciliary ganglion without synapsing.^217,218 Loss of these, if detected, would be incorrectly interpreted as evidence of corneal cholinergic innervation.

Acetylcholinesterase Histochemistry

The assumption has been made that acetylcholinesterase is a marker of cholinergic neurons. However, it can be found by biochemical assay in all cells. Laties and Jacobowitz²¹⁹ identified cholinergic innervation of the iris sphincter and ciliary body on the basis of cholinesterase stains. They were probably correct, but the ubiquitous distribution of the enzyme reduces the specificity of this approach.

Clear Vesicles

Nomura and Smelser²²⁰ identified clear vesicles in the nerve terminals of iris dilator muscle, iris sphincter muscle, and trabecular meshwork. They assumed that these contained acetylcholine and were cholinergic. Although acetylcholine is stored in clear vesicles, it is not as certain that all clear vesicles contain acetylcholine.

NICOTINIC DRUGS

Direct-Acting Nicotinic Agonists

This class of drugs does not play a role in ophthalmology.

Indirect-Acting Nicotinic Agonists

Cholinesterase inhibitors increase cholinergic activity in both muscarinic and nicotinic structures. With two exceptions, these drugs are used by the ophthalmologist for their muscarinic effects. One exception is their use in the diagnosis of myasthenia gravis.

Intravenous edrophonium (Tensilon) and, to a lesser extent, intramuscular neostigmine²²¹ have been the cholinesterase inhibitors used most frequently.

Edrophonium (Fig. 1) is a short-acting cholinesterase inhibitor. It carries a positive charge, enabling it to bind with the negatively charged “anionic” site of acetylcholinesterase. The bulky cyclic structure of edrophonium physically blocks the approach of acetylcholine to the “esteradic” site. In addition, electrostatic hydrogen bonding of the hydroxyl group of edrophonium causes inactivation of the esteradic site. Acetylcholine hydrolysis is inhibited because it must compete with edrophonium for both the “anionic” and “esteradic” sites. Edrophonium's binding to both enzyme sites is weak and short lived.

The side effects of intravenous edrophonium are the result of its action on muscarinic structures (bowel, bladder, and heart). They include abdominal cramping, urinary incontinence, bradycardia, and cardiac arrest.^222,223 However, the diagnostic value of edrophonium in myasthenia gravis resides in its nicotinic action: the striated musculature contracts more vigorously. Therefore, the side effects of edrophonium can be prevented without interfering with the drug's diagnostic efficacy by simultaneously administering a muscarinic blocking agent, such as atropine. This could be done as follows: Two syringes are used. One contains saline; the other contains atropine, 0.4 mg, and edrophonium, 10 mg. Neither the patient nor the physician evaluating the response knows which of the two solutions is being injected. The nurse gives one syringe to the physician for injection. Three tenths of the volume is injected rapidly, and the response is evaluated during the next minute. If this is well tolerated, the remainder is injected and the response is evaluated. The contents of the second syringe are then injected in the same manner. Only after the patient and physician have committed themselves to an evaluation of the relative efficacy of the two syringes does the nurse identify which contained the edrophonium.

Several different parameters have been suggested for evaluating the edrophonium response. The objective improvements in ocular and lid motility have been of primary interest to the ophthalmologist. Glaser and co-workers^224,225 used tonography to document the increased extraocular muscle tonus. They reported a mean 2-mmHg increase in intraocular pressure within 1 minute of injecting 10 mg edrophonium in 15 myasthenic patients. All 15 had unilateral, but not always bilateral, responses. The maximum elevation was 5 mmHg. The response could occur in less than 10 seconds, but the mean response interval was approximately 20 seconds²²⁶ to 35 seconds.²²⁷ Although there were no false-positive results in the 10 control patients in Glaser and co-workers' study, false-positive results can occur.^227,228 Wray and Pavan-Langston²²⁸ found that the intraocular pressure increase was greater in myasthenic patients without ophthalmoplegia than in those with ophthalmoplegia.

Kornbleuth and colleagues²²⁹ used electromyographic recordings of the extraocular muscles when testing with edrophonium. In their small series, only patients with myasthenia gravis showed an increase in electrical activity. Campbell and associates²²⁷ used electronystagmography to study the effect of edrophonium on the fatigue induced by optokinetic nystagmus. A beneficial response occurred in 50% of 17 myasthenia gravis patients given edrophonium injections. There were no false-positive reactions among 18 normal subjects. However, these authors found the test to be of only limited value because of the marked variations in amplitude and rate of optokinetic nystagmus among subjects.

Edrophonium increases the peak saccadic velocity in myasthenic patients but decreases it in normal controls.²³⁰ The use of topical cholinesterase inhibitor drops in the treatment of ocular myasthenia gravis has been suggested,²³¹ but their value is not well documented.

Cholinesterase inhibitors are also used for their nicotinic effects in the treatment of pediculosis of the lashes. The cholinesterase inhibitors are pesticides and kill by producing respiratory arrest. Cholinesterase inhibitors used primarily for their intraocular effects are relatively ineffective pesticides. Physostigmine 0.1% in peanut oil and 0.25% ointment kill the adult form of the crab louse (Phithirus pubis), but the nits are more resistant and must be removed with forceps.^232,233 Malathion is more effective and relatively safe because it is rapidly inactivated by human serum carboxylesterases. Within 5 minutes of exposure, 100% of lice are killed, and after 10 minutes of exposure 96% of eggs will not hatch. Two problems encountered when using malathion around the eyes are that commercial preparations contain a high (e.g., 78%) alcohol content and the drug gives off a foul-smelling sulfhydryl degradation product.

Demodex folliculorum is another pest infesting the lids. It is quite resistant to ophthalmic preparations. It can survive 3 or more days in 0.125% echothiophate, 0.25% demecarium, or 0.1% diisopropyl fluorophosphate.²³⁴

Nicotinic Antagonists

Two classes of nicotinic blocking agents exist: nondepolarizing antagonists (e.g., curare) and depolarizing antagonists (e.g., succinyldicholine).

NONDEPOLARIZING ANTAGONISTS. Neuromuscular Blockade. Duke-Elder and Duke-Elder²³⁵ showed that acetylcholine and nicotine caused contraction of dog extraocular muscles and that curare, but not atropine, could block these responses. Muscarine was subsequently shown to have a very weak contractile effect that could be blocked by atropine.²³⁶ Until the work of Katz and Eakins,²³⁷ it was believed that the ocular muscles were more sensitive to the blocking effects of nondepolarizing agents, such as curare and gallamine, than were the peripheral skeletal muscles.²³⁸ Katz and Eakins found that the reverse seemed true (e.g., a systemic dose of curare that paralyzed the cat anterior tibial muscle produced only a 50% block of the twitch response of the superior rectus). Their work was supported by Sanghvi and Smith.²³⁶ Systemic administration of nondepolarizing relaxants, such as alcuronium,²³⁹ atracurium,^240,241 fazadinium,²⁴² metocurine, and pancuronium,²⁴³ is associated with a lowering of intraocular pressure. Some of these have a rapid onset and short duration of action, which makes them useful as induction agents for intubation. These drugs also differ in their cardiovascular effects. For example, alcuronium and curare lower arterial blood pressure, whereas pancuronium increases blood pressure and heart rate.²⁴⁴

An effective neuromuscular block with atracurium or vecuronium lasts approximately 30 minutes, making these agents intermediate in their duration of action. Atracurium, unlike the shorter-acting depolarizing antagonist succinyldicholine, undergoes rapid ester hydrolysis at physiologic pH independent of serum butyrylcholinesterase activity. Large doses of atracurium have been suggested as a substitute for succinyldicholine induction because the former does not produce extraocular muscle contraction.^245,246 Atracurium 0.5 mg/kg did not produce a significant rise in intraocular pressure in subjects under steady-state nitrous oxide general anesthesia, but succinyldicholine 1 mg/kg did, from a mean baseline of 5.6 mmHg to 13.2 mmHg. Vecuronium seems to act in a manner similar to that of atracurium.^247,248

Mivacurium, at a dose of 0.15 mg/kg, has a slower onset of action (approximately 2.5 minutes) than succinyldicholine. At this dose, mivacurium's striated muscle block lasts approximately 16 minutes. Mivacurium, unlike atracurium and vecuronium, is inactivated primarily by butyrylcholinesterase.

Traction on the extraocular muscles during general anesthesia produces slowing of the heart. As stated earlier, pancuronium produces a tachycardia, which is protective. Strabismic children under general anesthesia who received pancuronium had heart rates that dropped from 120 ± 17 beats per minute to 94 ± 25 beats per minute.²⁴⁹ Patients receiving atracurium had pulse rates that fell from 86 ± 20 beats per minute to 34 ± 22 beats per minute, and 40% developed an additional arrhythmia besides the bradycardia. From a cardiac viewpoint, pancuronium has advantages in strabismus surgery.

Ganglionic Blockade. Transmission in sympathetic ganglia (e.g., superior cervical ganglion) and parasympathetic ganglia (e.g., ciliary ganglion) is primarily nicotinic. Several investigators have looked at ocular effects that might be related to partial blockade at this level. Gallamine²⁵⁰ and pancuronium²⁵¹ administration have been associated with a lowering of intraocular pressure. This may have been due, in part, to a reduction in systemic blood pressure. Intubation for general anesthesia produced tracheal irritation and a reflex increase in blood pressure. When throats were not anesthetized with topical anesthesia, the intraocular pressures 5 minutes after gallamine injection remained at baseline; when throats were anesthetized, the intraocular pressures were below baseline. The reductions in intraocular pressures produced by pancuronium were significant but were not dose related with the use of 0.01 to 0.08 mg/kg. Trimetaphan, a short-acting ganglionic blocking agent, administered intravenously produced reductions in intraocular pressure from preoperative values of 13 to 17 mmHg to intraoperative values of 3 to 4 mmHg as the systolic blood pressure fell to 60 mmHg.²⁵²

Hexamethonium and pentamethonium, 2 mg/kg, given intramuscularly to conscious subjects caused a mean maximum fall in intraocular pressure of 9 mmHg.²⁵³ The decline was present within 30 minutes of injection and persisted for 3 hours. This effect was attributed to a blockade of sympathetic ganglia and a resultant lowering of systemic blood pressure. Barnett²⁵³ treated hypertensive patients for 9 to 11 weeks with these drugs. He reported an improvement in the hypertensive retinopathy. However, these drugs produced an orthostatic hypotension that was disabling, which limited their use.

Retrobulbar tetraethylammonium injection in human subjects has produced mydriasis.²⁵⁴ The pupil remained dilated longer than 6 hours and did not constrict after topical physostigmine. Ciliary ganglion blockade also produced cycloplegia.^255,256 Decreased tearing has been attributed to blockade of the sphenopalatine ganglion after systemic administration of tetraethylammonium.²⁵⁵

Based on the assumption that sympathetic superior cervical and stellate ganglia fibers innervate and constrict the retinal arterioles, ganglionic blocking agents have been used in the treatment of central retinal artery occlusions, in the hope that the vessels would dilate and the embolus pass to a more distal arterial branch. However, there is little evidence that they are beneficial, and it can be argued that they may further reduce the perfusion blood pressure to the eye.

Botulinum Toxin. Botulinum toxins are produced by the anaerobic bacteria Clostridia botulinum. Each of eight different strains produces an immunologically distinct form of the toxin: A, B, C₁, C₂, D, E, F, and G. Four, A, B, D, and E, have been shown to block cholinergic function. They bind to unmyelinated cholinergic nerve terminals and prevent the release of quantal acetylcholine.^257,258 Death occurs by paralysis of the respiratory muscles; doses as low as 1 ng per kilogram body weight can be lethal in humans.²⁵⁹

Botulinum toxins are zinc-dependent proteases that attack proteins linking the acetylcholine-containing vesicles to the synaptic axon membrane. Calcium is no longer able to trigger a functional release of the transmitter.²⁸ The structure of botulinum A toxin has been fully sequenced.²⁶⁰ Type A botulinum toxin attacks a soluble 25-kd cytoplasmic attachment protein, SNAP-25,^29,261,262 abolishing quantal (i.e., functional) acetylcholine exocytosis; some nonquantal acetylcholine release does persist. The toxin does not block the neuron's action potential, the synthesis or storage of acetylcholine, the vesicle number, or calcium entry.²⁶³ The initial binding site of botulinum A toxin is a sialic acid-containing site on the external neuron surface; this attachment can be antagonized by lectins.²⁶⁴ Once inside the neuron, botulinum toxin can no longer be neutralized by antitoxins.

Type A botulinum toxin is more effective at nicotinic neuromuscular junctions than at nicotinic ganglion synapses or parasympathetic neuromuscular junctions. It has been used to treat essential blepharospasm, blepharospasm associated with facial dyskinesias, hemifacial spasm, nonparalytic and thyroid ophthalmopathy-induced strabismus, paralytic strabismus, nystagmus, corneal exposure, and entropion.^265–278

The toxin is injected into the muscle to be rendered paretic or subcutaneously in the adjacent area. The effect is dose dependent. Multiple small doses (e.g., 0.001 to 0.003 μg; 2.5 to 7.5 U) or fewer but larger doses may be given. The onset of effect is within 48 hours, and the maximum effect may take 5 to 6 days to develop. The results are usually transient and average 2 to 3 months in duration.

Histologic examination of orbicularis muscle fibers after prolonged treatment of blepharospasm failed to show morphologic changes. However, the motor neurons had many unmyelinated axon sprouts, most of which ended blindly; those ending on muscle fibers were able to elicit new and multiple motor end plates at previously nonsynaptic sites.^279,280

In general, botulinum A treatment is of little value in strabismus in which the eye muscle movements are restricted, and it is of less value than traditional surgery in comitant deviations.²⁸¹ Retrobulbar injections, used to treat nystagmus and oscillopsia, may be of temporary value depending, in part, on the amount and duration of ptosis produced.^282–285 Complications occur from local spread of toxin to adjacent muscle fibers and from an inability to accurately predict the magnitude of the effect. Ptosis is one of the most common complications caused by local spread of toxin.²⁸⁶ Ptosis, partial or complete, can at times be of therapeutic value (e.g., before recovery from Bell's palsy).^287–289 Deliberate ptosis can be achieved by injecting botulinum A toxin along the superior orbital roof, approximately 25 mm behind the superior orbital rim.

Clinical signs of systemic toxicity have not been reported, but there is evidence of subtle systemic effects (e.g., abnormal electromyographic recordings in arm muscle fibers of patients receiving 0.005 μg of toxin).²⁹⁰ Neurologists treat dystonias with relatively large doses of botulinum A toxin; these may produce only subtle distant electromyographic changes but are likely to elicit neutralizing antibodies that limit the toxin's usefulness.^291–293 A distinction should be made between non-neutralizing antibodies, which may have no clinical significance, and neutralizing antibodies.²⁹⁴

DEPOLARIZING ANTAGONISTS. Extraocular Muscle Effects. The depolarizing antagonists, such as succinyldicholine, decamethonium, and hexacarbacholine (Fig. 2), paralyze striated voluntary muscles throughout the human body but have a different effect on the extraocular muscles. The extraocular muscles respond with a sustained contraction. This was first noted by Hofmann and Holzer,²⁹⁵ who reported that the contraction of the extraocular muscles caused an increase in human intraocular pressure. This observation was supported by Lincoff and co-workers.^296,297 The maximum intraocular pressure reported is 55 mmHg.²⁹⁸

The shortening effect and increased muscle tension created by succinyldicholine have been measured in humans.²⁹⁹ The drug always produced contraction in the horizontal and vertical recti muscles. The results in the oblique muscles were variable.

The explanation is that there are two types of extraocular muscle fibers. In the center of each muscle, typical striated muscle fibers, with a single myoneural junction (en plaque fibers), are most numerous. These are paralyzed by succinyldicholine. However, the surface layers contain fibers with multiple myoneural junctions. These are the “en grappe” fibers. Their innervation has the appearance of a bunch of grapes. It is these fibers that respond to succinyldicholine with a sustained contraction.^300–302 The strength of contraction depends on the dose of succinyldicholine and the number of multiply innervated fibers. In sheep superior oblique muscle, succinyldicholine produces only 7% of the force generated by maximum electrical stimulation of the entire muscle.³⁰³ In cats, up to one third of the whole muscle force has been generated by succinyldicholine.³⁰⁴ Succinyldicholine causes a partial reduction in the resting membrane potential and a localized contraction in the area around each myoneural junction.³⁰⁵ Because these junctions are found along the entire length of the en grappe fiber, most of each fiber is depolarized and shortened.³⁰⁶ However, unlike en plaque fibers, which are capable of generating action potentials, the en grappe fibers are believed to respond to both neuronal impulses and succinyldicholine with slow, graded contractions (i.e., they are unable to conduct action potentials).³⁰⁷ This may explain why Kornbleuth and colleagues²²⁹ found a dissociation between ocular muscle electrical activity and the elevation of intraocular pressure. They performed simultaneous tonography and electromyography on subjects given succinyldicholine. They expected to find that the increase in intraocular pressure was proportional to the degree of extraocular muscle electrical activity. Electromyography was performed on antagonistic vertical or horizontal recti of the same eye in subjects receiving 5, 10, 15, or 20 mg succinyldicholine. These doses were too small to produce apnea. A 2- to 4-mmHg increase in intraocular pressure occurred in patients under general anesthesia, and a 5- to 14-mmHg increase occurred in conscious volunteers. The duration, but not the magnitude, of the ocular pressure elevation appeared to be dose related. The rise in intraocular pressure elevation occurred just as the electrical activity of the extraocular muscle was reduced. The inability of the en grappe fibers to conduct action potentials may explain this dissociation.

Curare, but not atropine, blocked the sustained contraction produced by succinyldicholine in animal studies.³⁰⁸ Katz and Eakins²³⁷ found evidence that the en plaque fiber response to depolarizing antagonists was quantitatively different for eye muscles and skeletal muscles (e.g., the dose of succinyldicholine needed to depress the twitch response of the superior rectus muscle was greater than that needed to block skeletal muscles). This difference was less when decamethonium was used.

Each of the six human extraocular muscles contains multiply innervated and singly innervated fibers in about the same proportions: in the periphery, 92% ± 10% are multiply innervated, and in the center, 33% ± 12% are multiply innervated.³⁰⁹ The contractile effect of succinyldicholine on the en grappe fibers is generally considered undesirable. However, Hannington-Kiff³¹⁰ suggested that the anesthesiologist use the resultant change in interlimbal distance to monitor the action of succinyldicholine. The interlimbal distance in 35 patients under general anesthesia before succinyldicholine injection was 52.9 mm ± 4.3 mm. Ninety seconds after injection, this distance had decreased to 49 mm ± 5.5 mm. One minute after the return of spontaneous respirations, the interlimbal distance was near baseline values, 51.4 mm ± 3.3 mm. The duration of succinyldicholine stimulation of en grappe fibers and the duration of succinyldicholine paralysis of en plaque fibers appeared similar. Taylor and associates³¹¹ also found that the intraocular pressure returned to baseline with the onset of spontaneous respirations. Pandey and co-workers³¹² studied the time course of the ocular pressure elevation in 34 patients given succinyldicholine in a dose of 1.5 to 2 mg/kg. An increase in intraocular pressure was present in less than 1 minute, peaked in 2 to 4 minutes, and returned to baseline in 6 minutes.

Succinyldicholine stimulation of the extraocular muscles results in a variable amount of enophthalmos; as much as 3.25 mm has been reported.³¹³ The conjunctival vessels dilate,³¹⁴ and the forced-duction test may suggest a mechanical restriction for up to 20 minutes.³¹⁵

Mindel and colleagues³¹⁶ theorized that the basic deviation of the eyes was produced by the balance of forces exerted by the multiply innervated muscle fibers. They used intravenous succinyldicholine to stimulate these fibers and to remove the influence of the singly innervated fibers, which were paralyzed. Under general anesthesia, esotropic patients became esotropic when 2 mg/kg was injected. Exotropic patients became exotropic.³¹⁷ Despite this qualitative success, the measured amount of drug-induced ocular deviation did not agree closely with that in the conscious patient. These authors believed that this incongruity was due to A and V pattern effects, that is, in conscious patients the eyes could be maintained in the primary position by fixation, whereas in anesthetized patients the eyes could move vertically as well as horizontally. Lingua and associates³¹⁸ have reported similar findings. In addition, they found a correlation between the ocular position induced by succinyldicholine immediately after strabismus surgery and the 1-week postoperative result.

Intraocular Muscle Effects. Abramson³¹⁹ used ultrasound to measure anterior chamber depth and lens thickness in human subjects given 1 mg/kg succinyldicholine intravenously. Succinyldicholine caused a rapid increase in anterior chamber depth and a decrease in lens thickness. This effect was present within 20 seconds, was maximal in 45 to 210 seconds, and disappeared in 3 to 5 minutes. Abramson attributed this effect to a relaxation of accommodation. However, there was no change in the diameter of the pupil. It is unlikely that succinyldicholine would decrease the activity of only one of the two parasympathetic structures innervated by the ciliary ganglion.

Side Effects. Succinyldicholine may produce a bradycardia. This is usually associated with multiple injections. However, asystole has been reported after a single dose.³²⁰ Succinyldicholine and, to a lesser degree, its first hydrolysis product, succinylmonocholine, are responsible for this cardiac effect.³²¹

Succinyldicholine is inactivated by esterase hydrolysis. The apnea produced by respiratory muscle paralysis may be prolonged in patients with genetically³²² or acquired reduced serum cholinesterase activities.^323,324 Chronic systemic absorption from topical cholinesterase inhibitor eyedrops can result in such an effect.³²⁵ In one report, a reduction of serum cholinesterase activity 62% below normal from echothiophate eyedrops permitted endotracheal intubation using a total of only 9.5 mg succinyldicholine.³²⁶ Decamethonium, because it is excreted unmetabolized, is not dependent on the level of serum cholinesterase activity for termination of its effect.

There are at least four alleles, at a single locus, that control the type of serum cholinesterase made by the liver: normal, dibucaine resistant, fluoride resistant, and silent genes. Approximately 1 in 4000 in the population will develop prolonged apnea after succinyldicholine injection. This is the incidence of a person having both genes abnormal. Approximately 1 in 400 in the population will be a normal dibucaine-resistant heterozygote and will develop a shorter but still significantly prolonged apnea. Other factors reducing serum cholinesterase activity are hepatic disease,³²⁷ malnutrition, anemia, severe dehydration, late pregnancy,³²⁸ and systemic absorption of cholinesterase inhibitors. In one study, 5% of a general surgical population had decreased serum cholinesterase activity.³²⁹

The sustained contraction of the extraocular muscles after succinyldicholine injection poses a threat if there is a preexisting perforating laceration or if an incision is to be made into the eye (e.g., cataract extraction). Expulsion of the intraocular contents can result from the steady squeezing of the eye muscles. Lincoff and colleagues²⁹⁷ mentioned three personal communications linking the use of succinyldicholine to this complication. There are several ways it can be avoided: After succinyldicholine injection, the intraocular pressure is monitored with a tonometer until it returns to normal. Use of depolarizing nicotinic antagonists is avoided. Alternatively, a pharmacologic antagonist can be used to prevent the effect. With regard to this last suggestion, cholinesterase inhibitors are not of value. Although they reverse the action of nondepolarizing muscle relaxants (e.g., curare), they prolong and enhance the action of depolarizing drugs.³³⁰ Theoretically, pretreatment with small doses of curare or succinyldicholine itself might prevent succinyldicholine-induced ocular muscle contraction. However, the results are conflicting.^331,332 Macri and Grimes³³³ found that prior curare injection prevented ocular hypertension in cats given succinyldicholine. Because both drugs competed for the same receptor, multiple doses of succinyldicholine overcame the curare effect. Miller and associates³³⁴ found that 1 mg/kg succinyldicholine produced a mean intraocular pressure increase of 8.5 mmHg in 10 control subjects. Parenteral gallamine, 20 mg, or curare, 3 mg, administered 3 minutes before succinyldicholine, prevented this effect in both glaucomatous and normal subjects. However, when Meyers and co-workers³³⁵ studied the value of curare, 0.09 mg/kg, or gallamine, 0.3 mg/kg, given 3 minutes before 1 to 1.5 mg/kg succinyldicholine, they found that ocular pressure elevations still occurred (e.g., in 10 patients with a mean intraocular pressure of 13 ± 1 mmHg, curare did not prevent an elevation to 24 ± 1.3 mmHg). Pancuronium, 0.14 mg/kg, also was ineffective.³³⁶ Katz and colleagues³³⁷ found that hexafluorenium, which is both a nondepolarizing (curarelike) blocking agent and a plasma cholinesterase inhibitor, in doses of 0.4 mg/kg, prevented the rise in intraocular pressure from succinyldicholine, 0.3 mg/kg. Hexafluorenium was effective only if it was injected before succinyldicholine, not after it.

Perhaps the preceding inconsistencies result from the multiplicity of factors that can affect the intraocular pressure (e.g., depth of anesthesia, serum O₂ and CO₂ levels, venous pressure, and tracheal stimulation). Barbiturates, given to induce anesthesia, can reduce muscle endplate currents.³³⁸ When thiopental was injected 2 minutes before succinyldicholine, 1 mg/kg, there was no significant increase in ocular pressure.³³⁹ Macri and Grimes³³³ disinserted all the extraocular muscles in cats. This reduced, but did not prevent, an increase in intraocular pressure after succinyldicholine. Taylor and associates³¹¹ reported that in 25 of 29 patients, the blood pressures 1 minute after succinyldicholine injection were elevated. These may have contributed to the short-term elevations in ocular pressure.

The blood pressure elevations could be because succinyldicholine raises blood catecholamine levels. Succinyldicholine, but not curare, increased plasma norepinephrine levels immediately after injection.³⁴⁰ Succinyldicholine, 1 mg/kg, produced a peak norepinephrine elevation 3 minutes after injection, raising levels from a mean of 301 pg/mL at baseline to 647 pg/mL. The increase had disappeared by 10 minutes. The elevation in epinephrine levels was less marked and became insignificant by 2 minutes.

Wynands and Crowell³⁴¹ found that 50% of patients given succinyldicholine had a rise in intraocular pressure. In 3 of 18 subjects, the elevation was more than 10 mmHg. However, endotracheal intubation alone produced a rise of more than 10 mmHg in 10 of 23 subjects. Lewallen and Hicks³⁴² and Craythorne and co-workers²⁹⁸ reported conflicting results in subjects without intubation. The former found that injection of 40 mg succinyldicholine did not raise human intraocular pressure; the latter found that continuous infusion of 0.1% to 0.2% succinyldicholine did.

A most intriguing study was that of 15 patients having uniocular enucleations.³⁴³ These subjects were anesthetized with 3 to 4 mg/kg thiopental intravenously and maintained with halothane or isoflurane and nitrous oxide/oxygen. No premedications were used. All six extraocular muscles were severed from the eye to be enucleated. Intraocular pressures were then obtained bilaterally. Succinyldicholine 1.5 mg/kg was administered and the intraocular pressures were measured every 30 seconds for 5 minutes. The systolic and diastolic blood pressures were not significantly different before and 90 seconds after succinyldicholine administration, but the intraocular pressures were significantly and maximally elevated bilaterally at this time. Surprisingly, there was no significant difference in the mean ± SE intraocular pressure rise between those eyes with severed muscles and those eyes with muscle insertions intact. The presuccinyldicholine injection intraocular pressures of the normal and muscle-severed eyes were, respectively, 15.1 ± 1 mmHg and 16.1 ± 1 mmHg. At 90 seconds after succinyldicholine injection, the respective intraocular pressures were 25.2 ± 1.6 mmHg and 24.7 ± 1.8 mmHg. Perhaps an increase in venous blood pressure produced these bilateral results, but the apparent lack of an extraocular muscle effect is inexplicable.

MUSCARINIC DRUGS

Direct-Acting Muscarinic Agonists

GENERAL CONSIDERATIONS. The ocular effects of this class of drugs include reduction in intraocular pressure; stimulation of the iris sphincter, producing miosis; and stimulation of the ciliary muscle, producing accommodation. Examples of direct-acting muscarinic drugs are muscarine, pilocarpine, aceclidine (3-acetoxyquinuclidine), arecoline, and acetyl β-methylcholine (methacholine, Mecholyl) (Figs. 3 through 5). In high concentration, pilocarpine also stimulates choline acetyltransferase synthesis of acetylcholine,³⁴⁴ but this is probably of little clinical significance.³⁴⁵

Isopilocarpine is a naturally occurring stereoisomer of pilocarpine present in very small amounts in most preparations of the drug.³⁴⁶ Isopilocarpine has one tenth the binding affinity of pilocarpine for bovine ciliary muscle and has very little pharmacologic activity.

Carbachol (carbamylcholine) is both a direct-acting muscarinic agonist and a direct-acting nicotinic agonist. In addition, it has indirect agonist activities (i.e., it is a cholinesterase inhibitor). This last action results from the NH₂ substitution on the acetyl group of carbachol. It markedly reduces the molecule's susceptibility to hydrolysis by acetylcholinesterase but does not prevent carbachol from competing with acetylcholine for the anionic and esteradic sites of acetylcholinesterase. This interference with acetylcholine hydrolysis results in a further increase in cholinergic activity. Aceclidine, too, is slightly cholinesterase resistant and has weak anticholinesterase activity.

Pilocarpine's structure is quite different from that of acetylcholine. Either of the nitrogen atoms in the imidazole ring can become positively charged and bind to the anionic receptor site. Pilocarpine does not have the classic muscarinic agonist structure of acetylcholine (N-C-C-O-C-C). However, pilocarpine's interatomic spacing (N-C-C-C-C) may mimic it.³⁴⁷ Evidence exists that the intrinsic muscarinic activity of pilocarpine is less than that of acetylcholine and carbachol.^348,349 However, additional factors play a role in determining clinical efficacy. These are primarily pharmacokinetic. For example, acetylcholine and acetyl β-methylcholine are hydrolyzed by tissue esterases and may not reach the site at which their action is desired. Inhibiting tissue cholinesterases increases the efficacy of both drugs. Arecoline, aceclidine, and pilocarpine are tertiary amines and can penetrate the corneal epithelium more readily than the positively charged quaternary amines such as acetylcholine, carbachol, and acetyl β-methylcholine. This correlates with the lipid solubilities of these drugs. As measured with toluene: pH 6.5 buffer, the partition coefficient of arecoline is 0.1; pilocarpine, 0.033; aceclidine, 0.002; and carbachol, <0.0001.³⁵⁰ The pHs of eyedrops and tears alter the proportions of nonionized drug (e.g., the ability of arecoline and oxotremorine to reverse mydriasis increases as a function of the pHs of their solutions).³⁵¹ Drugs that are ionized at all pHs, such as carbachol, are concentration dependent, not pH dependent. The poor corneal penetration of carbachol resulted in Clarke³⁵² finding marked variability in its ability to lower intraocular pressure. O'Brien and Swan³⁵³ performed a corneal massage through the lids of human subjects. This allowed a 0.09% carbachol solution to be as effective a miotic as a 1.5% to 4.5% carbachol solution. Topical anesthetics and surfactants also aided penetration (e.g., the addition of 0.03% benzalkonium to 1.5% carbachol produced significant accommodation for 48 hours).

Accurate estimates of corneal penetration by pilocarpine have been impaired by the use of tritiated molecules. Asseff and co-workers³⁵⁴ gave anesthetized monkeys eyedrops of 1% to 8% tritiated pilocarpine. They reported approximately 3% corneal penetration at 5 minutes. Chrai and Robinson,³⁵⁵ studying rabbits, found similar levels after applying unpurified tritiated pilocarpine but only 0.13% corneal penetration at 5 minutes after applying purified material. Maximum penetration occurred at 20 minutes and was 0.18%. At 2 hours, the aqueous humor concentration was 0.03% of the administered dose. These authors attributed the previous higher values to tritium exchange between pilocarpine and its solvent during storage. Penetration of the tritiated solvent resulted in falsely high estimates of corneal penetration. Krohn and Breitfeller³⁵⁶ applied two drops of 2% pilocarpine HCl (approximately 2000 μg) to corneas of patients under general anesthesia; the pilocarpine concentration in the aqueous humor was not more than 5 μg/mL.

Pilocarpine can be administered from continuous-release membranes (Ocusert) made of polymerized ethylenevinylacetate. Ideally, the low-dosage form (Ocusert P20) releases 20 μg/h and the high-dosage form (Ocusert P40) releases 40 μg/h. However, Armaly and Rao³⁵⁷ found that the in vitro release rates from membranes supposedly dispensing 20 μg/h, 50 μg/h, and 80 μg/h were two and one half to three times their projected rates during the first 7 hours of use. Between 7 and 24 hours, the release rates remained slightly elevated, but within the next 24 hours they fell to slightly below their projected levels and continued to decline. After 3 to 4 days, the release rates were 78%, 88%, and 81% of those expected. These results may explain why the glaucoma of two of Armaly and Rao's patients was controlled for the first 2 days of membrane use but not thereafter.

Pilocarpine has also been applied as a polymer emulsion^358,359 and as a gel. Both provide prolonged drug delivery to the eye and, compared with drops, longer duration of action. The gel is available commercially as 4% pilocarpine HCl in a viscous aqueous acrylic gel vehicle; this concentration of pilocarpine approaches the binding limit of the polymer used to make the gel. A single daily application of the gel has a hypotensive effect for 24 hours. For the first 18 hours, this effect is indistinguishable from that elicited by 4% pilocarpine eyedrops given four times a day in terms of both pressure and pupillary diameter.³⁶⁰ For hours 18 to 24, the drops are more effective.³⁶¹

Pathology can alter pharmacokinetics and drug response. Patients with such diverse pathology as atopy and trisomy 21 are said to be more sensitive to muscarinic agonists and antagonists.^362,363

Patients with intraocular inflammation inactivate pilocarpine. Schonberg and Ellis³⁶⁴ found that the primary aqueous humor of humans and rabbits did not inactivate pilocarpine, but serum and secondary aqueous did. Ten percent of 500-μg pilocarpine was inactivated by 200 μL of secondary aqueous humor in 1 hour. Inactivation could be prevented by heating serum to 60°C for 10 hours, by dialysis, or by the addition of some (e.g., penicillamine and ethylenediaminetetraacetic acid), but not all, chelating agents. Inactivation could not be inhibited by anticholinesterase drugs. Rabbit serum was more active against pilocarpine than human serum. Only at high pilocarpine concentrations were human ocular tissues (cornea, iris-ciliary body, lens, retina, and choroid) more active than the corresponding rabbit ocular tissues.³⁶⁵ Lee and colleagues³⁶⁶ found that pigmented rabbit corneas inactivated pilocarpine much more effectively than did albino rabbit corneas.

OCULAR PHARMACOLOGY Iris Sphincter Muscle. ANIMAL STUDIES. Pilocarpine has less intrinsic muscarinic activity than the endogenous transmitter acetylcholine. As a result, Swan and Gehrsitz³⁶⁷ found that rabbits given topical 0.25% physostigmine or 0.1% DFP had less miosis if 4% pilocarpine was previously administered. If pilocarpine were injected into the cornea after maximum DFP miosis, a transient dilatation occurred. These results were consistent with pilocarpine-acetylcholine competition for the same receptors.

Miosis in mammals is associated with M₃ receptor activation and increased phospholipase C activity.^368,369 Feedback inhibitory prejunctional muscarinic receptors on sympathetic neurons of the iris dilator muscle seem to be of a different subtype.¹⁹⁹ In rabbits, these prejunctional receptors respond to methacholine, oxotremorine, muscarine, and carbachol by inhibiting norepinephrine release; atropine can block this effect.³⁷⁰

Denervation supersensitivity of the rat iris sphincter is not associated with a change in muscle fiber resting potential but is associated with increased sensitivity to calcium.^371,372 Thus, supersensitivity could be due to an increased release of intracellularly bound calcium and/or an increased influx of extracellular calcium.

Bito and Banks³⁷³ administered DFP drops chronically to monkeys and then discontinued the drug. They observed several interesting pupillary phenomena, one of which was a diminished response to direct-acting muscarinic agonists during the recovery period. For at least 3 days after discontinuing DFP, the pupils responded to light but did not respond to carbachol or pilocarpine. Similar results were obtained in the cat.³⁷⁴ These results may have been produced from an acquired subsensitivity of the sphincter muscarinic receptors (i.e., a subsensitivity produced by the prior high acetylcholine concentrations). Another possibility is that the greater intrinsic agonist activity of acetylcholine, compared with that of pilocarpine and carbachol, became manifest. Other data from this laboratory,³⁷⁵ confirmed by Claesson and Barany,³⁷⁶ suggested that an alteration of muscarinic receptor sensitivity had occurred. Exposure of feline eyes in vivo to continuous light resulted in subsensitivity to pilocarpine, whereas light deprivation resulted in increased sensitivity. After 7 days in ambient light of 1200 lumens/m², the irides became insensitive to pilocarpine. Surprisingly, subsensitivity to carbachol or acetyl β-methylcholine could not be demonstrated in vitro. Hemicholinium (which reduces acetylcholine synthesis) injected intravitreally in vivo resulted in increased sensitivity to administered muscarinic agonists. Barany³⁷⁷ reported that chronic administration of pilocarpine drops did not produce subsensitivity in experimental animals challenged with intravenous pilocarpine. However, after prolonged treatment in the form of continuous-release membranes, pilocarpine did produce subsensitivity.

Perhaps the explanation for the aforementioned phenomena is miosis from peptide transmitters. There is evidence that the neurotransmitter substance P, identified in trigeminal nerve fibers, produces miosis.³⁷⁸ Mandahl³⁷⁹ has suggested that, in the rabbit, the miosis elicited by cholinesterase inhibitors and pilocarpine is primarily the result of release of substance P from trigeminal nerve endings in the iris. According to this theory, continuous application of cholinesterase inhibitors and pilocarpine would deplete the stores of substance P, resulting in a reduced response, whereas the acetylcholine release by light-stimulated parasympathetic fibers would remain intact.

HUMAN STUDIES. Lowenstein and Loewenfeld³⁸⁰ and Newsome and Loewenfeld³⁸¹ studied the iris using pupillography. Drops of 1% to 2% pilocarpine were given to volunteers without ocular disease. The onset of miosis, maximum miosis, and duration of miosis varied greatly from person to person. Maximum miosis occurred in 25 to 45 minutes, was maintained for 1 to 3 hours, and required 24 to 36 hours for full recovery. The pupillary reflex to light persisted unless miosis was maximal. However, during the recovery phase, the light response was markedly diminished. When submaximally effective concentrations of pilocarpine (e.g., 0.1%) were followed by submaximal concentrations of a cholinesterase inhibitor (e.g., 0.1% physostigmine), the results were additive.

Ogle and associates³⁸² and Morgan and co-workers³⁸³ also used pupillography to study miosis in normal subjects. They reported latencies after 1% pilocarpine of 9.8 ± 0.9 minutes. The latencies for 0.75% carbachol and 0.25% physostigmine were 13.7 ± 2.2 minutes and 13.9 ± 1 minutes, respectively. The rates of miosis and amounts of maximum miosis were less for carbachol than for pilocarpine and physostigmine, yet all three drugs required about the same time to produce their maximum effect. The times required for recovery to 50% of the predrop pupil diameters were: carbachol, 164 ± 46 minutes; pilocarpine, 271 ± 109 minutes; and physostigmine, 456 ± 28 minutes. The latency of mydriasis from 0.5% tropicamide (Mydriacyl) was similar to the latency of miosis from pilocarpine. The changes in pupillary diameter due to light stimuli during the latency periods of both pilocarpine and tropicamide were similar; the amounts and maximum rates of pupil constriction after onset of action also were similar.

After the short-term application of various direct-acting muscarinic agonists, the response produced by the light reflex was reduced.³⁸⁴ This may have represented competition between residual exogenous agonists, with weaker intrinsic activity, and the endogenous agonist acetylcholine, with more potent intrinsic activity. The light reflex required approximately 24 hours to recover after 2 drops of 2% pilocarpine, 7 hours to recover after 10 drops of 0.2% arecoline hydrobromide, and 24 hours to recover after 10 drops of 0.2% aceclidine hydrochloride.

Ciliary Muscle. ANIMAL STUDIES. Tornqvist^385–387 studied accommodation in monkey eyes. Intramuscular pilocarpine over a dose range of 0.2 to 2 mg/kg appeared to have equal affinity for iris and ciliary muscle. The lowest dose caused 2 diopters (D) or less of accommodation, and the highest dose always caused more than 12 D of accommodation. One monkey was given higher doses of pilocarpine, and the myopia paradoxically decreased: 2 mg/kg produced 20.3 D of accommodation, 5 mg/kg produced 11.7 D of accommodation, and 50 mg/kg produced 8 D of accommodation. Similarly, small doses of systemic carbachol, 0.005 to 0.1 mg/kg, produced increasing accommodation, but larger doses, 0.1 to 0.5 mg/kg, produced decreased accommodation. This phenomenon could not be reproduced by using high concentrations of topical muscarinic agonists. Retrobulbar anesthesia did not prevent this paradoxical effect, indicating that central nervous system and ganglionic effects were not the cause. Intravenous atropine prevented, or reversed, the paradoxical effect. A topical dose of pilocarpine that produced half the maximum accommodative effort was 100 times more than that needed to produce half the maximum miosis. Because systemically administered pilocarpine did not exhibit this difference, it was concluded that pharmacokinetic factors resulted in more drug reaching the iris than reached the ciliary muscle after topical application.

Barany³⁸⁸ studied subsensitivity of the monkey's ciliary muscle after a single drop of topical carbachol given unilaterally. For approximately 1 week, the accommodative response in the treated eye was less than that in the untreated eye after systemic administration of pilocarpine, carbachol, or bethanechol. The reasons for this effect were not clear, but the author did not believe the answer was a reduction in the number of muscarinic receptors or a reduction in their binding characteristics.

In monkeys, the loss of accommodation that occurs with aging is not associated with a reduction in the number of ciliary muscle muscarinic receptors.³⁸⁹

The agonist aceclidine is less effective in producing accommodation than in increasing miosis and outflow facility. Studies in monkey eyes found that M₃ receptors predominate in ciliary muscle, iris sphincter muscle and trabecular meshwork (i.e., the differences in pharmacologic responses could not be attributed to different muscarinic receptor subtypes).^390,391

After ciliary ganglionectomy, monkey ciliary muscle supersensitivity to pilocarpine was associated with a 12% to 84% reduction in muscarinic receptors.³⁹² The magnitude of this seemingly paradoxical finding is difficult to explain simply by attributing it to the loss of prejunctional muscarinic receptors on nerve endings.

HUMAN STUDIES. Ciliary body thickness was measured in volunteers by the use of ultrasound.³⁹³ Two hours after applying a drop of 2% pilocarpine, the increase in mean thickness was 0.06 mm. Anterior chamber depth was measured after single and multiple drops of 2% or 4% pilocarpine.³⁹⁴ All subjects showed a decrease in anterior chamber depth after a single drop of pilocarpine. This shallowing remained significant for the 6 hours that measurements were made. When compared with the untreated eye, a single drop of 2% pilocarpine produced a mean maximum shallowing of 0.09 mm that occurred 3 hours after instillation; 4% pilocarpine produced a mean maximum shallowing of 0.07 mm that occurred 4.5 hours after instillation. Pilocarpine (2%), every 6 hours, produced a mean maximum anterior chamber narrowing of 0.12 mm. This was not significantly different from the single-drop effect. One week after chronic therapy was discontinued, the mean depth of the treated eye was not significantly different from that of the control eye. Abramson and colleagues³⁹⁵ confirmed that the anterior chamber shallowing effect from 2% pilocarpine, as well as the increase in lens thickness, was similar after single-drop and chronic (10-day) therapy.

Hallden and Henricsson³⁹⁶ produced with-the-rule astigmatism in 10 young men by asymmetrically contracting the ciliary body. This was achieved by applying a cotton pledget containing 10% pilocarpine to the temporal bulbar conjunctiva for 3 minutes, creating 0.75- to 2 D myopia with an axis between 0° and 20°.

Intraocular Pressure. The site at which muscarinic drugs act to lower intraocular pressure is a subject of controversy. Their effect is believed to be local, although an occasional report raises the possibility of a systemic effect (e.g., Willetts³⁹⁷ reported that normotensive subjects given unilateral pilocarpine drops had a bilateral decrease in ocular pressure). However, subjects with a mean plasma level of 2.9 ng/mL, delivered by skin patches, did not have a significant reduction in intraocular pressure.³⁹⁸

The different mechanisms of action that have been proposed include a decrease in aqueous humor secretion; an increase in removal of aqueous humor by active transport; an increase in removal of aqueous humor by passive trabecular outflow (from increased iris sphincter tone, increased ciliary muscle tone, or decreased episcleral venous pressure); and an increase in removal of aqueous humor by way of nontrabecular routes.

AQUEOUS HUMOR SECRETION. The data from different laboratories are inconsistent. Becker³⁹⁹ found that pilocarpine drops increased rabbit aqueous humor formation in vivo. Green and colleagues⁴⁰⁰ reported that acetylcholine and pilocarpine produced an increase in rabbit aqueous humor secretion in vitro. Macri and Cevario⁴⁰¹ administered pilocarpine intra-arterially to enucleated cat eyes and also found an increase in aqueous humor secretion; however, outflow facility was increased, producing an overall reduction in intraocular pressure. Berggren^402,403 found the opposite results. He used in vitro thinning of rabbit ciliary processes as a measure of secretion. Pilocarpine 10^-9 mol/L, physostigmine 10^-7 mol/L, acetylcholine 10^-5 mol/L, and carbachol 10^-5 mol/L inhibited secretion. The therapeutically inactive stereoisomer of pilocarpine, isopilocarpine, did not block secretion. Atropine 10^-7 mol/L alone had an inhibitory effect⁴⁰² and also prevented the more potent pilocarpine effect.⁴⁰³ However, when intravenous pilocarpine, 2 mg/kg, was injected in vivo 5 minutes before the in vitro studies, there was no evidence of altered secretion.

Edwards and colleagues,⁴⁰⁴ using constant-pressure perfusion, measured the effect of unilateral topical 4% pilocarpine on monkeys under general anesthesia. Pilocarpine caused an increase in aqueous humor production. However, Walinder and Bill⁴⁰⁵ found in vervet monkeys that infusion of pilocarpine 10^-4 mol/L into the anterior chamber produced a reduction in aqueous humor formation of approximately 1 μL/min; atropine infusion 3 × 10^-5 mol/L abolished the pilocarpine effect.

Barsam⁴⁰⁶ found that the hypotensive effect of pilocarpine lasted longer than the duration of increased outflow facility in human subjects. He believed that pilocarpine was producing a reduction in aqueous humor secretion. However, fluorophotometry of normotensive eyes has provided evidence that pilocarpine has only a minor effect on aqueous humor formation, which is to increase secretion by approximately 14%.⁴⁰⁷ This may or may not be true for the glaucomatous eye as well.

Muscarinic agonists might alter aqueous humor secretion by altering ciliary blood flow. Stjernschantz and Bill⁴⁰⁸ studied cholinergic control of uveal blood flow in anesthetized animals. Stimulation of the intracranial third nerve altered the blood flow to the iris and ciliary body but not to the retina and choroid. In the cat, the ciliary body blood flow was increased; in the rabbit, it was decreased; and in the monkey there was no statistically significant effect. In all three species, the iris blood flow was decreased by third-nerve stimulation. Intravenous atropine blocked this iris effect in all three species and prevented the increased ciliary body blood flow in cats.

AQUEOUS HUMOR ACTIVE TRANSPORT. Large vacuoles have been observed in the endothelium of the inner wall of Schlemm's canal. It has been suggested that they represent the result of active transport of aqueous humor. Holmberg and Barany⁴⁰⁹ found that monkey eyes treated with pilocarpine had a reduced number of vacuoles. Lutjen-Drecoll⁴¹⁰ placed 20 μg pilocarpine hydrochloride into the anterior chambers of monkeys and then studied the eyes with electron microscopy. Large vacuoles were rarely encountered. Grierson and associates⁴¹¹ studied the enucleated eyes of eight patients with melanomas. Pilocarpine drops were given before their removal. These were compared with 11 control eyes but not in a masked manner. There were more than twice the number of large vacuoles in the pilocarpine-treated eyes. However, the authors' results from baboon eyes led them to conclude that the increased vacuolization was not due to a direct effect of pilocarpine on the endothelium of Schlemm's canal. Instead, they attributed the vacuoles to a secondary effect from increased trabecular flow.⁴¹²

INCREASE IN PASSIVE AQUEOUS HUMOR OUTFLOW. Most investigations have dealt with this mechanism of muscarinic action. Edwards and co-workers⁴⁰⁴ found that topical 4% pilocarpine caused more than a doubling in monkey eye outflow facility, from 0.41 ± 0.04 μL/min/mmHg to 1.09 ± 0.14 μL/min/mmHg. Atropine alone had no effect. Barany⁴¹³ found that pilocarpine, administered topically or injected into the anterior chamber, increased vervet monkey facility of outflow. Two components appeared to be present, because atropine, injected intravenously or intraocularly, reversed part of the increase in 3 to 5 minutes and the remainder in approximately 30 minutes. Lutjen-Drecoll⁴¹⁰ studied electron micrographs of monkey eyes and found that the trabecular pore area in contact with the inner wall of Schlemm's canal correlated, after pilocarpine administration, with the outflow facility.

In humans, the hypotensive effect of pilocarpine is usually, but not always, associated with an increase in outflow facility. Outflow facility, accommodation, and pilocarpine-induced accommodation decrease with age. However, the pilocarpine-induced increase in outflow facility and reduction in intraocular pressure were found not to decrease with age.⁴¹⁴ Pilocarpine, using gonioscopy to evaluate the drug's effect, seems to produce a bowing inward of the trabecular meshwork toward the anterior chamber and away from the canal of Schlemm.⁴¹⁵

Gaasterland and colleagues⁴¹⁶ found that normotensive subjects given a single drop of 4% pilocarpine had, within 2 hours, a reduction in mean intraocular pressure from 14.6 to 11.2 mmHg and an increase in outflow facility from 0.24 to 0.40 μL/min/mmHg. Willetts⁴¹⁷ studied the effect of 2% pilocarpine, three times a day for 4 to 5 days, in normal eyes. Although intraocular pressures declined, he found no significant alterations in outflow facility. Krill and Newell⁴¹⁸ found that normal and glaucomatous eyes responded to four-times-a-day pilocarpine therapy with a reduction in pressure that was proportional to the initial pressure (i.e., the higher the initial intraocular pressure, the greater the absolute amount of pressure reduction). However, the percent declines were similar: 8% to 38% in normal eyes and 12% to 40% in glaucomatous eyes.

Although pilocarpine increased the initial outflow facility, there was no correlation between the magnitude of the increase and the fall in pressure. In two normal and five glaucomatous eyes there was little or no outflow facility increase. Grant,⁴¹⁹ Becker and Friedenwald,⁴²⁰ and Harris and Galin⁴²¹ reported similar findings. Barsam⁴⁰⁶ and Flindall and Drance⁴²² reported a temporal dissociation between the effects of muscarinic agonists on outflow facility and intraocular pressure. Barsam⁴⁰⁶ found that the effect on outflow facility reached a maximum at 2 hours and then dissipated so rapidly that it appeared that pilocarpine was acting through a decrease in aqueous humor formation. Flindall and Drance⁴²² reported a dissociation in the opposite direction. Five hours after a single unilateral drop of 0.75% or 1.5% carbachol, the intraocular pressure reduction was no longer significant. However, the outflow facility remained increased, by 24.5% and 24.1%, respectively, compared with that of the control eye. Kronfeld⁴²³ found that a single drop of pilocarpine produced, in general, more lowering of intraocular pressure than could be explained by the increase in outflow facility. This was true whether the drop was given to normotensive subjects or to glaucoma patients. In the latter, therapy had been discontinued 1 week before the test drop. Lieberman and Leopold⁴²⁴ reported that aceclidine lowered intraocular pressure without increasing outflow facility. Investigations of the mechanism by which increased outflow facility occurs have dealt with the following actions:

IRIS SPHINCTER CONTRACTION. The hypotensive action of muscarinic agonists could result from miosis if the iris base pulled on the scleral spur and widened the trabecular meshwork pores. However, there is little evidence to support such a mechanism. In glaucomatous beagles, the miosis from muscarinic agonists is maximal at 45 to 120 minutes postinstillation, after which recovery begins; however, the hypotensive action is greatest 2 to 5 hours after instillation. Thorburn⁴²⁵ gave normal human eyes a 4% pilocarpine drop. The pupillary diameter was minimum at 30 minutes, but the intraocular pressure was not significantly decreased. O'Brien and Swan³⁵³ reported that three eyes with surgical sphincterotomies continued to respond to 1.5% carbachol with a reduction in intraocular pressure below 20 mmHg. Mapstone⁴²⁶ found that 2% pilocarpine followed by 10% phenylephrine lowered intraocular pressure in normal eyes from a baseline of 14.9 mmHg to 13.7 mmHg and increased outflow facility from 0.25 μL/min/mmHg to 0.33 μL/min/mmHg; measurements were made 90 minutes postinstillation. When a second drop of phenylephrine was given 2.5 hours after the first, there was an insignificant decrease in intraocular pressure and the outflow facility increased from 0.33 μL/min/mmHg to 0.38 μL/min/mmHg. Bito and Merritt⁴²⁷ dissociated the miotic effect of pilocarpine from the hypotensive effect in monkeys. Chronic treatment with echothiophate resulted in pilocarpine producing full miosis and an increase in intraocular pressure instead of the pretreatment reduction.

CILIARY MUSCLE CONTRACTION. Flocks and Zweng⁴²⁸ investigated the possibility that ciliary muscle contraction pulled on the scleral spur and widened the trabecular pores. Pilocarpine drops were given to one eye of monkeys and atropine to the other. The monkeys were killed with intraperitoneal pilocarpine. Eyes were examined histologically and by gonioscopy. These investigators believed the histologic appearance was that of trabecular lamellae being stretched and separated in the eyes receiving pilocarpine. Gonioscopy was stated to be too insensitive to allow evaluation. When Jampel and Mindel⁴²⁹ unilaterally stimulated the nucleus of accommodation in the midbrain of macaque monkeys, up to 10 D of accommodation was achieved. Maintenance of this marked accommodation for 100 seconds did not alter intraocular pressure relative to the control eye; however, Armaly and Burian⁴³⁰ found that accommodation in normal human subjects caused a more rapid decline in the intraocular pressure during tonography. Thorburn⁴²⁵ reported that the maximum increase in outflow facility in normal human eyes occurred 30 to 60 minutes after a 4% pilocarpine eyedrop and coincided with the refractive change that occurred from ciliary muscle contraction. However, the temporal course of the ocular hypotensive effect did not coincide with either the outflow facility or the degree of accommodation. Abramson and colleagues^395,431 found that individual pilocarpine drops in glaucoma patients on chronic therapy produced maximum lens thickening and shallowing of the anterior chamber in 1 hour. By 2 hours, these effects were almost gone, but the hypotensive action was maintained far longer. Grierson and associates⁴¹¹ studied human eyes given pilocarpine before enucleation. The scleral spurs in treated eyes appeared to be pulled more posteriorly, widening the scleral sulcus by a mean value of approximately 15° but narrowing the lumen of Schlemm's canal.

Kaufman and Barany⁴³² disinserted and recessed surgically the ciliary muscles of monkey eyes. Not only did this not result in elevated intraocular pressures, but the intraocular pressures remained lower than in control eyes during the more than 1-year period after disinsertion. Perhaps cyclodialysis clefts had been created. However, because outflow facility did not increase when intravenous and intraocular pilocarpine was injected 12, 33, and 56 weeks postoperatively, these authors believed that their model supported the ciliary muscle-scleral spur traction theory.

It has also been proposed that the pull of the ciliary muscle might act to lower intraocular pressure by pulling open a collapsed canal of Schlemm.^433,434

In final consideration of the ciliary muscle-scleral spur traction theory, it is interesting to note that one of the beneficial aspects of pilocarpine sustained-release therapy (Ocuserts) is that a dissociation is created between the degree of hypotension and the degree of induced myopia. Maximum hypotension occurs with minimal stimulation of the ciliary muscle. François and colleagues⁴³⁵ reported that the miosis, myopia, and shallowing of the anterior chamber were less with the Ocusert P20 than with 2% pilocarpine drops. The mean accommodative myopia was: age 20 to 40 years, 5.8 D with drops versus 0.8 D with Ocusert; age 40 to 60 years, 1.3 D with drops versus 0.3 D with Ocusert; and age greater than 60 years, 0.2 D with drops versus 0.0 D with Ocusert.

EPISCLERAL VENOUS PRESSURE. The aqueous humor collection channels eventually empty into the episcleral venous circulation. If the episcleral venous pressure were increased or decreased by muscarinic agonists, the rate of aqueous humor outflow would be decreased or increased, respectively. Wilke⁴³⁶ found that topical 2% pilocarpine produced a transient rise in intraocular pressure in normal human eyes. The elevation was 2 to 5.5 mmHg and lasted 20 to 40 minutes. It was followed by a reduction in intraocular pressure beginning 40 to 60 minutes postinstillation. The early period of pressure elevation was associated with dilatation of conjunctival and episcleral vessels and an increase in episcleral pressure of 3.5 to 5 mmHg lasting 9 to 17 minutes. Gaasterland and associates⁴¹⁶ gave normotensive human subjects a single drop of 4% pilocarpine and reported that 1 hour later, during the hypotensive phase, there was virtually no change in episcleral venous pressure (i.e., it was 9.3 ± 0.3 mmHg before pilocarpine and 9.5 ± 0.4 mmHg 1 hour after pilocarpine administration).

NONTRABECULAR OUTFLOW PATHWAYS. The pseudofacility seen on tonographic tracings may represent, at least in part, aqueous humor outflow by routes other than the trabecular meshwork. Bill and Walinder⁴³⁷ instilled 10^-4 mol/L pilocarpine unilaterally in monkey eyes. Uveoscleral flow was decreased, compared with that of the control eye, whereas drainage by way of the trabecular system was increased and intraocular pressure was elevated by a mean value of 2.6 ± 0.7 mmHg. These authors theorized that contraction of the ciliary muscle caused the diminished uveoscleral flow. Gaasterland and co-workers⁴¹⁶ found that topical 4% pilocarpine, given to normotensive humans, increased pseudofacility during the hypotensive response.

Lacrimation. The lacrimal gland receives a parasympathetic innervation that may control secretion. In familial dysautonomia there is decreased lacrimation, and it has been suggested that this innervation may be deficient. However, topical solutions of 2.5% and 20% acetyl β-methylcholine failed to increase lacrimation in 12 and 6 dysautonomic subjects, respectively, although miosis occurred. Failure was defined as less than a 5-mm increase in Schirmer paper strip wetting. The phenotypically normal parents of these patients also failed to respond with increased lacrimation.⁴³⁸ Smith and colleagues⁴³⁹ injected acetyl β-methylcholine intravenously. The effect on lacrimation was determined by observation only. Increased lacrimation occurred in normal controls at infusion rates of 2.5 to 11.7 μg/kg/min. In patients with dysautonomias, increased lacrimation occurred at infusion rates that were lower, 0.4 to 2.8 μg/kg/min. This confirmed the earlier observation by Kroop,⁴⁴⁰ who produced lacrimation in patients with dysautonomias with subcutaneous acetyl β-methylcholine. These results were consistent with the finding that acetyl β-methylcholine was ineffective topically because it could not reach the appropriate lacrimal receptors.

DeHaas⁴⁴¹ reported three patients with unilateral sensory and/or motor paresis of the lacrimal reflex. Pilocarpine, 7 to 10 mg parenterally, produced lacrimation on the damaged side. In only one subject was the effect on the contralateral normal side stated; it also was increased.

THERAPEUTICS. Open-Angle Glaucoma. A number of studies found that lowering the intraocular pressure preserved the vision of patients with chronic open angle glaucoma.^442–448

Drance and Nash⁴⁴⁹ gave 1%, 2%, 4%, and 8% pilocarpine hydrochloride in 0.5% hydroxypropyl methylcellulose, pH 4.5, to 12 ocular hypertensive patients without field loss. Intraocular pressures were recorded every 15 minutes for the first hour and hourly thereafter for 7 more hours. Each concentration of pilocarpine was given as a single drop a week apart, and the order of concentrations was randomized except for the 8% solution, which was always given last. During the first hour, there was usually a pressure rise. A statistically significant intraocular hypotensive effect occurred between hours 1 and 2 and lasted for nearly 8 hours. The reduction from 1% pilocarpine was about half maximal and was of shorter duration. Although higher concentrations gave dose-related increased reductions in ocular pressure, the 8% solution was no more effective than the 4% soultion. However, a maximum increase in outflow occurred in some patients at 8%; in some patients, the maximum outflow effect occurred at 1% or 2%. Harris and Galin⁴²¹ also found that the 8% pilocarpine solution was no more effective than the 4% solution in lowering ocular pressure.

Worthen⁴⁵⁰ studied the effect of pilocarpine, 2% or 4%, four times a day, on the diurnal pressure curve of open-angle glaucoma patients. Pilocarpine not only lowered the intraocular pressure by a mean value of 9 mmHg but it also flattened the diurnal curve by decreasing the maximum fluctuation from 18.5 mmHg before treatment to 8.5 mmHg during treatment. The maximum pressure-lowering effect of pilocarpine occurred between 9 AM and 6 PM. Pratt-Johnson and colleagues⁴⁵¹ found that 4% pilocarpine four times a day was less effective in controlling the diurnal variation than 0.06% echothiophate twice a day. The mean variation while on pilocarpine was 12 mmHg and that while on echothiophate was 7 mmHg.

Rothkoff and associates⁴⁵² investigated whether the hypotensive response to a single drop of 2% pilocarpine would predict the efficacy of chronic treatment. There were three parts to their study: (1) one drop of 2% pilocarpine hydrochloride was instilled at 8 AM and the pressure was measured hourly for 4 hours; (2) all subjects were subsequently admitted and administered pilocarpine every 4 hours beginning at 6 AM and ending at 10 PM, with intraocular pressures measured hourly from 7 AM to 11 PM; and (3) all subjects were discharged on 2% pilocarpine and followed at monthly intervals for 3 months. Rothkoff and co-workers found that they could not predict whose intraocular pressure would be controlled consistently below 24 mmHg but that they could, from the single-drop response, predict whose would not be controlled. These authors also found that hypotensive efficacy did not correlate with initial intraocular pressure. Subjects responding to the single drop with a pressure below 24 mmHg had baseline pressures of 30.7 ± 5.2 mmHg; these were not significantly different from those of the failures, which were 33.4 ± 4.6 mmHg. Data from other studies leave doubt that a single application of pilocarpine will predict the efficacy of chronic therapy. Van Hoose and Leaders⁴⁵³ found that pilocarpine accumulated in the cornea (i.e., the cornea acted as a reservoir). This might explain why Harris and Galin⁴²¹ did not find significant dose-related differences in the hypotensive action of single drops of 1% to 10% pilocarpine. These were given to 25 open-angle glaucoma patients off therapy for 3 weeks. However, on chronic therapy, 4% and 8% concentrations were more efficacious than a 1% concentration. The mean percent decline in ocular pressure at 90 minutes from a single drop was 1% pilocarpine, 18.25%; 4% pilocarpine, 18.78%; and 10% pilocarpine, 20.85%. One week of chronic therapy produced mean declines of 1% pilocarpine, 17.48%; 4% pilocarpine, 26.76%; and 8% pilocarpine, 29.07%. These authors noted that there seemed to be two populations of responders to the single drops. One group responded with a marked fall in intraocular pressure (i.e., 34%) to a single drop of 1% pilocarpine; this group responded similarly to the 10% solution (i.e., with a 35% decrease in pressure). The other population responded relatively poorly to 1% pilocarpine (8% decline) and to the higher concentrations as well (16% decline).

In subjects with maximum intraocular pressures of 24 to 37 mmHg, Flindall and Drance⁴²² found that a single drop of carbachol had a maximum hypotensive effect at 4, 4, and 2 hours for 0.75%, 1.5%, and 3% solutions, respectively. The two lower concentrations produced similar maximum responses. Recovery was complete at 6 hours for 0.75% carbachol and at 8 hours for 1.5% carbachol, whereas a near maximal response remained 8 hours after the 3% solution. The absolute reduction in intraocular pressure was greater, in millimeters of mercury, for eyes with higher baseline values. However, when expressed as percent reduction, there was no difference.

There appeared to be no consistent advantage to substituting carbachol for pilocarpine. When 26 patients on 2%, 4%, or 6% pilocarpine four times a day were changed to 3% carbachol three times a day, 18 had either no additional benefit or an elevation in intraocular pressure. The remaining 8 patients had an additional mean 4.3-mmHg reduction in intraocular pressure.⁴⁵⁴

Barsam⁴⁰⁶ found that open-angle glaucoma subjects taken off 2% pilocarpine therapy for 48 hours and given a single drop of 2% pilocarpine or 0.03% or 0.06% echothiophate had maximum pressure reduction 2 to 4 hours after the pilocarpine and 4 to 6 hours after the echothiophate. The maximum hypotensive effect was similar for all three solutions, but the duration of effect was greater for the echothiophate solutions.

Romano⁴⁵⁵ used a double-masked cross-over study to compare 3 weeks of three-times-a-day instillation of 2% aceclidine and 2% pilocarpine in open-angle glaucoma patients. Both were effective, and the differences between them were insignificant with regard to hypotensive effect, outflow facility effect, and miosis.

The hypotensive action of a sustained-release form of pilocarpine (Ocusert) was simulated using eyedrops every 3 minutes.⁴⁵⁶ The minimum effective dose was between 10 and 30 μg/h. Armaly and Rao³⁵⁷ found that membranes dispensing 50 μg/h were more effective in open-angle glaucoma therapy than were 20-μg/h membranes. This was confirmed by Drance and associates.⁴⁵⁷ Release rates higher than 40 μg/h were no more effective.^357,457

Intraocular pressure control 4 hours after insertion of a 20-μg/h membrane was similar to that 4 hours after instillation of a 2% pilocarpine eyedrop.³⁵⁷ The duration of hypotensive efficacy of this membrane was at least 1 week; that of a membrane releasing 40 μg/h was at least 10 days.

The concept of “tolerance” to muscarinic agonists and the beneficial effects of “rest therapy” are occasionally encountered. For example, Clarke³⁵² found that subjects no longer responsive to pilocarpine therapy would respond after a pilocarpine-free period. Inactive solutions and patient noncompliance were not considered in explaining this phenomenon. Pharmacologically, it is difficult to understand why tolerance would develop so infrequently and why reinstitution of therapy would not result in a reappearance of tolerance. Sustained-release therapy (Ocusert) provides continuous exposure to pilocarpine and would be expected to lead to an increased incidence of tolerance, but this has not been reported (e.g., Worthen and co-workers⁴⁵⁸ found that membrane therapy for periods up to 8 months was as effective as pilocarpine drops).

Muscarinic agonists and β-sympathetic receptor blocking drugs have an additive effect. A solution containing 4% pilocarpine and 0.5% timolol administered twice a day produced a greater mean ± SD reduction in intraocular pressure (9.2 ± 5.1 mmHg) in glaucoma patients than did 4% pilocarpine four times a day (5.6 ± 3.6 mmHg) or 0.5% timolol twice a day (7.5 ± 5 mmHg).^459–461 Similar results have been reported for combination solutions of pilocarpine-carteolol.

Transient Ocular Hypertension. Yttrium-aluminum-garnet laser capsulotomies and argon laser trabeculoplasties are often followed by a transient elevation in intraocular pressure. Pilocarpine (4%) drops administered immediately after laser therapy reduce the frequency and degree of these complications.^462,463 In pilocarpine-treated trabeculoplasties, the mean intraocular pressure 2 hours after the procedure was 22 mmHg, whereas in the untreated group the mean was 28 mmHg.

Twenty-five glaucoma patients never treated pharmacologically had laser trabeculoplasties preceded 1 hour before by two drops of 2% pilocarpine; their mean ± SD maximum increase in intraocular pressure in the first 2 hours post-treatment was 2.4 ± 4.4 mmHg.⁴⁶⁴

The use of viscoelastic substances during cataract surgery is often associated with a transient postoperative elevation in intraocular pressure. Intracameral carbachol (0.01%), given in volumes of 0.1, 0.25, or 0.5 mL at the conclusion of surgery, was more effective than saline in controlling the intraocular pressure during the following 48 hours.⁴⁶⁵ Most effective was the 0.5-mL volume. This was confirmed in another study in which 8 of 30 eyes receiving saline had intraocular pressures exceeding 25 mmHg at 6 hours and 4 eyes exceeded 25 mmHg at 18 hours.⁴⁶⁶ None of the 30 patients receiving 0.5 mL 0.01% intracameral carbachol exceeded 25 mmHg at 6 hours, and only 1 of these 30 eyes exceeded 25 mmHg at 18 hours. One inch of 4% pilocarpine gel, placed in the cul-de-sac at the conclusion of surgery, was also effective.⁴⁶⁷ Intracameral acetylcholine (1%) was effective at 3 and 6 hours after surgery but not at 9 hours and 24 hours.⁴⁶⁸

Closed-Angle Glaucoma. Pilocarpine drops have been used to break attacks of acute closed-angle glaucoma. The rationale behind their frequent success is that the lax iris base is pulled taut and away from the trabecular meshwork. A similar rationale has been used to explain their value in preventing future attacks. Of Winter's⁴⁶⁹ patients with unilateral closed-angle glaucoma, 68% had involvement of the second eye. Although this occurred from hours up to 18 years after involvement of the first eye, most had contralateral involvement within 5 years. In 20 patients who maintained a continuous pharmacologic miosis, only 40% had a contralateral attack. In 8 patients maintained on intermittent therapy, 88% had a contralateral attack. In 19 patients without any therapy, 89% had a contralateral attack.

However, a narrow line exists between prophylactic miosis and provocative pupillary block. Thus, Lowe⁴⁷⁰ reported that two patients developed a contralateral attack shortly after stopping prophylactic pilocarpine but a third patient developed acute glaucoma just after starting pilocarpine. He believed 2% pilocarpine would be less likely to produce pupillary block than higher concentrations and suggested that, because most spontaneous attacks seemed to occur during the night, prophylactic drugs should be taken after the evening meal and again at bedtime. He cautioned that the miosis produced by physostigmine was more intense than that of pilocarpine and more likely to produce pupillary block. This is supported by Bain's⁴⁷¹ results, in which 21% of 137 patients treated prophylactically with physostigmine or pilocarpine developed acute closed-angle glaucoma. Only 17% of 103 patients using pilocarpine had an attack, whereas 32% of 34 patients using physostigmine had an attack. Fifty-four percent of an untreated control group of 39 patients developed a second attack.

Mapstone⁴⁷² used pilocarpine as part of a provocative test to predict which eyes with narrow angles were likely to develop acute narrow-angle glaucoma. He observed that the increase in ocular pressure after provocative testing tended not to occur when the pupil was widely dilated but, rather, when the pupil was mid-dilated. He believed that mid-dilation was the optimal position for maximizing the two crucial factors: trabecular block by the iris base and pupillary block by the iris sphincter. He found that the combination of a muscarinic agonist, pilocarpine, with an adrenergic agonist, phenylephrine, gave a greater incidence of positive provocative testing than did muscarinic atagonists. In 70% of 49 subjects with prior acute closed-angle glaucoma, pressure elevations of 8 mmHg or more were obtained within 2 hours of administering phenylephrine plus pilocarpine. Only 33% of subjects responded to 0.5% tropicamide. The response was reversed within 90 minutes of injecting acetazolamide, 500 mg, intravenously and applying one drop of 2% pilocarpine topically. However, this provocative test with a combination of pilocarpine and phenylephrine lacks sensitivity. The test was repeated at yearly intervals in the contralateral eyes of 104 patients who previously presented with unilateral acute narrow-angle glaucoma.⁴⁷³ Mean follow-up was 10 ± 5.7 years. Of 56 eyes with consistently negative tests, 14 developed acute glaucoma and 8 developed chronic or subacute glaucoma (i.e., 40% of eyes with consistently negative tests developed some form of closed-angle glaucoma).

Accommodative Esotropia. The near synkinesis consists of accommodation, convergence, and miosis. Decreased accommodative effort results in decreased convergence. This can be achieved by muscarinic agonists: pharmacologic stimulation of the ciliary muscle results in lens accommodation and reduces the need for central nervous system input. This rationale also applies to a nonpharmacologic technique of therapy, convex lenses. By reducing the neurogenic component of accommodation, both forms of therapy reduce accommodative convergence.

Pilocarpine is effective in reducing accommodative esotropia.⁴⁷⁴ Whitwell and Preston⁴⁷⁵ used it for a limited period beginning 2 to 3 days after strabismus surgery. Pilocarpine (1%), instilled in each eye three times a day, was gradually tapered over an 8-week period and then discontinued. Its use allowed fusion to develop, with or without the aid of orthoptics, if a residual accommodative component to the esotropia remained. Used in this manner, therapy was of permanent value in only those patients capable of stereopsis.

The longer-acting indirect muscarinic agonists have largely replaced the direct agonists in the therapy of accommodative esotropia. The former's use will be discussed in the section on cholinesterase inhibitors.

Intraoperative Miosis. Ray,⁴⁷⁶ Harley and Mishler,⁴⁷⁷ and Rizzuti⁴⁷⁸ injected acetylcholine into the anterior chamber after cataract extraction to protect the vitreous face and prevent iris incarceration into the surgical wound. Harley and Mishler⁴⁷⁷ dilated the eyes of patients preoperatively with 5% homatropine and 10% phenylephrine. Approximately 0.5 mL of 0.1%, 0.2%, 0.5%, or 1% acetylcholine was used. All concentrations were effective but short lived. The use of α-chymotrypsin did not affect the miosis. The stability of acetylcholine solutions was dependent on the pH and temperature. At 25°C and pH 5, the half-life of acetylcholine solutions was 14 years; at pH 7, 50 days; at pH 8, 5 days; and at pH 10, 1 hour.⁴⁷⁹ The pH of the commercially available 1% acetylcholine solution (Miochol) was approximately 8.5.

Douglas⁴⁸⁰ compared the efficacy of 1% acetylcholine with that of 0.01% carbachol (Miostat). In eyes dilated preoperatively with 5% homatropine and 10% phenylephrine, the miotic response to either solution injected into the anterior chamber was similar at 2 and 5 minutes. Several hours after surgery, the reduction in pupillary diameter, 4.4 ± 0.3 mm, was significantly greater in the eyes receiving carbachol than in the eyes receiving acetylcholine (2.9 ± 0.5 mm). Twenty-four hours after surgery there was no significant difference.

Drugs that inhibit prostaglandin synthesis provide increased mydriasis during intraocular surgery. Preoperative treatment with 0.03% flurbiprofen eyedrops did not prevent the miotic response 1, 2.5, and 5 minutes after intracameral acetylcholine.⁴⁸¹

Reversal of Pharmacologic Mydriasis. α-Adrenergic agonists, such as 10% phenylephrine and 1% hydroxyamphetamine, produced mydriasis that was reversed by 1% pilocarpine.⁴⁸² The mydriatic drops were given 65 to 70 minutes before pilocarpine. The pilocarpine-induced mean recovery time after phenylephrine was 22 minutes and that after hydroxyamphetamine was 14 minutes; however, 1% pilocarpine produced myopia as well as overcorrected mydriasis (i.e., miosis occurred).

The material deposited in exfoliation syndrome may impede iris movement. In 20 patients with exfoliation syndrome, phenylephrine produced less dilation than it did in 20 age-matched controls, and there was less constriction from a 10-μL drop of 4% pilocarpine given to reverse this mydriasis.⁴⁸³

Variable results have been reported when pilocarpine was used to reverse the mydriasis of muscarinic antagonists. One study found that one drop of 1% pilocarpine administered 30 minutes after tropicamide dilation not only returned the pupil to baseline diameter 3.5 hours later but continued to produce constriction, resulting in an overcorrection.⁴⁸² However, another study of 23 volunteers found that one drop of 2% pilocarpine given unilaterally 30 minutes after bilateral tropicamide dilation was no more effective in reversing the mydriasis and cycloplegia 30, 120, and 180 minutes later than was the drop of saline placed in the other eye.⁴⁸⁴ Pilocarpine (1%), administered 72 minutes after homatropine dilation, was of little if any beneficial effect.⁴⁸² Mean pupil recovery time was 28 hours. Reversal of mydriasis from a combination of 0.1% dilute tropicamide and 1% hydroxyamphetamine was “easily produced” by 2% pilocarpine. ⁴⁸⁵ Recovery from the mydriasis produced by three drops of 0.5% cyclopentolate was reduced from less than 20 hours to less than 6 hours by 1% pilocarpine.⁴⁸⁶

Occasionally, a patient will present with a dilated fixed pupil. If the patient is a nurse, he or she will be especially upset because of the known associations with aneurysms and tentorial herniation. In this situation, however, a likely cause is inadvertent self-administration of an atropinic agent: the nurse's fingers were contaminated when atropine drops were administered to a patient. If inadvertent or deliberate self-administration is suspected, a 0.5% or 1% pilocarpine drop administered bilaterally will be diagnostic. The pharmacologically dilated pupil will not respond as rapidly or as markedly, if at all. However, a dilated pupil due to an acquired efferent (third nerve) defect will respond. Rarely, congenital defects, such as absence of the sphincter muscle, will produce a mydriasis unresponsive to pilocarpine.⁴⁸⁷

Reversal of Pharmacologic Cycloplegia. Muscarinic agonists have been used to reverse cycloplegia. Although cholinesterase inhibitors have been used for this purpose with some success,⁴⁸⁸ most of the experience is with pilocarpine. In patients who had an initial dose of two drops of 0.5% cyclopentolate, followed 5 minutes later by a third drop, 1% pilocarpine reduced recovery time from 24 hours to 6 hours.⁴⁸⁹ One drop of 1% pilocarpine allowed subjects 14 to 35 years of age to read 2 to 4 hours after receiving 1% tropicamide.⁴⁹⁰ Pape and Forbes⁴⁹¹ reported a retinal detachment after a single drop of 1% pilocarpine that was given to reverse mydriasis and cycloplegia.

Diagnosis of Supersensitivity. TONIC PUPIL (ADIE'S SYNDROME). In this entity the iris sphincter and ciliary muscle exhibit supersensitivity to direct-acting muscarinic agonists. The underlying pathology is believed to be degeneration of the postciliary ganglion fibers.⁴⁹² A similar pathology is believed to occur in the tonic pupils associated with dysautonomias, such as Shy-Drager syndrome. The earliest pharmacologic test of sphincter function used physostigmine.⁴⁹³ However, it was not until the work of Scheie⁴⁹⁴ that pharmacologic testing of tonic pupils became routine. Scheie demonstrated supersensitivity with acetyl β-methylcholine. The supersensitivity appeared to be a permanent phenomenon: Laties and Scheie⁴⁹⁵ reported that it persisted in a subject for 25 years. It was initially believed that a drop of 20% acetyl β-methylcholine would constrict all pupils but that a drop of 2.5% would constrict only a supersensitive pupil. Subsequently, 41% of normal pupils were found to constrict more than 0.2 mm after 2.5% acetyl β-methylcholine,⁴⁹⁶ whereas only 63% of patients with a clinical diagnosis of Adie's syndrome had a positive response.⁴⁹⁷ Ocular hypertensive patients, previously untreated, had significantly greater pupil constriction to 2.5% acetyl β-methylcholine than did age- and sex-matched controls.⁴⁹⁸ Concentrations of acetyl β-methylcholine as high as 25% did not always constrict normal pupils.⁴⁹⁹ This variability in responses is, at least in part, attributable to differences in drug corneal penetration.⁵⁰⁰ False-negatives can result from increased tearing and blinking.

False-positive tests can result from abnormalities in the corneal epithelium that increase drug penetration (e.g., patients with exophthalmos and blink defects may give positive results,^501,502 the interpretation of which must await repeated testing after the recovery of the corneal epithelium).⁵⁰³ For similar reasons, prior administration of eyedrops containing local anesthetics or surfactants should be avoided.

Despite these difficulties, the 2.5% acetyl β-methylcholine test remained in clinical use until the drug was no longer readily available for topical use. Another direct-acting muscarinic agonist was looked for, and found. Pilocarpine became heir to the acetyl β-methylcholine test even though the latter subsequently reappeared in a commercial preparation.

The pilocarpine test is best performed under conditions in which ambient lighting and accommodation are controlled, because both can affect pupil diameter. The pupil diameter can be evaluated by observation, photographs, or pupillography. A drop of test solution is instilled bilaterally. Thirty to 60 minutes after instillation, the pupillary diameters are reevaluated under the same conditions as before.

What concentrations of pilocarpine are optimal for demonstrating a tonic pupil? Pilocarpine 0.125% to 0.25% has been advocated even though the normal pupil will almost always constrict. Because the drops are instilled bilaterally, it does not matter if a submaximal constriction of the normal pupil is elicited. The relatively greater miotic response of the tonic pupil identifies it as supersensitive. Advocates of 0.0625% to 0.1% pilocarpine argue that tonic pupils can occur bilaterally and that relative miosis is, therefore, difficult to interpret. Instead, the less concentrated solutions should be used so that any constriction can be interpreted as evidence of supersensitivity. Unfortunately for this argument, not only will an occasional tonic iris not respond to a drop of 0.1% pilocarpine (i.e., there are false-negatives), but normally innervated irides may respond to 0.0625% (i.e., false-positives can occur). For example, 6 of 10 normal irides showed some constriction 30 minutes after a drop of 0.04% pilocarpine.⁵⁰⁴

Because 0.03125% to 0.05% concentrations of pilocarpine did not consistently constrict supersensitive irides, Cohen and Zakov⁵⁰⁵ and Young and Buski⁵⁰⁶ suggested using 0.0625% to 0.1% solutions. The 0.0625% pilocarpine solutions produced a mean 0.5-mm constriction in normal pupils and a mean 3.5-mm constriction in patients with Adie's syndrome. Pilley and Thompson⁴⁹⁷ and Bourgon and colleagues⁴⁹⁶ found that 85% of normal irides constricted more than 0.2 mm after 0.125% pilocarpine solutions. However, more constriction occurred in eyes with Adie's syndrome. This permitted the authors to make the correct diagnosis of supersensitivity 80% of the time. The mean constriction was 2.4 mm in normal eyes and 4.2 mm in supersensitive eyes.⁵⁰⁵ Hedges and Gerner⁵⁰⁷ advocated using a 0.25% pilocarpine concentration, whereas Young and Buski⁵⁰⁶ found 0.2% pilocarpine too potent for supersensitivity testing. In summary, the pharmacologic tests of iris sphincter denervation appear somewhat unreliable.

The relative pharmacologic supersensitivity of the ciliary muscle in Adie's syndrome has been examined. Single drops of 0.25% pilocarpine were instilled bilaterally, followed by a second set of drops 30 seconds later.⁵⁰⁸ An interocular difference of 1 D or more was considered significant. Seventy-three percent of patients with a clinical diagnosis of a unilateral lesion developed more myopia in the abnormal eye. An asymmetric increase in astigmatism occurred in one third of positive tests; this was attributed to sector supersensitivity of the ciliary muscle. However, pharmacologic testing appeared to be of little clinical value because 0.5 D or more of asymmetry in accommodation could be detected in 66% of Adie's syndrome patients simply by measuring the near point. Furthermore, the lens has lost almost all accommodative power by age 50, so neither pharmacologic nor near-point testing is of value in these older patients.

Ciliary Ganglion Denervation. Several investigators have examined whether damage to the oculomotor nerve fibers innervating the ciliary ganglion (i.e., preganglionic denervation) resulted in iris sphincter muscle supersensitivity to muscarinic agonists. Ponsford and colleagues⁵⁰⁹ found that oculomotor nerve palsies resulted in supersensitivity to 2% acetyl β-methylcholine. Fourteen normally innervated eyes developed a mean 0.25-mm constriction, whereas the contralateral eyes with oculomotor nerve palsies responded with a 0.96-mm constriction. In a second study, only 3 of 5 patients showed supersensitivity.⁵¹⁰ One potential confounding factor in interpreting pharmacologic testing is that aberrant regeneration of extraocular muscle nerve fibers to the ciliary ganglion can produce pupil diameter changes when eye movements are made.⁵¹¹ Jacobson⁵¹² applied two drops of 0.1% pilocarpine bilaterally. Supersensitivity was defined as being present if, at 30 minutes, either the eye with the oculomotor nerve paresis constricted 0.5 mm more than the contralateral eye or the involved eye became the more miotic of the two. Supersensitivity was present in 5 of 11 eyes with tumor compressions, 4 of 5 eyes with traumatic palsies, 2 of 2 eyes with congenital palsies, and 0 of 5 eyes with idiopathic pupil-involving palsies. Supersensitivity, when present, did not correlate with the degree of anisocoria.

TOXICITY AND SIDE EFFECTS. The clinical use of direct-acting muscarinic agonists has been relatively free of significant side effects and toxicities.

The inadvertent 2-mL subcutaneous injection of an ophthalmic preparation of 4% pilocarpine (80 mg) in a 39-year-old woman was not fatal. It produced diaphoresis, salivation, nausea, diarrhea, and dyspnea; these symptoms gradually cleared over a 4-day period.⁵¹³ However, deaths have been attributed to systemic pilocarpine toxicity.⁵¹⁴

O'Brien and Swan³⁵³ stated that systemic and local reactions to carbachol were rare. The eyeache and headache that occurred initially were usually gone by the third day of therapy. Direct-acting muscarinic agonists produce fewer side effects than cholinesterase inhibitors.⁴⁵¹ Drance and Nash⁴⁴⁹ used 1% to 8% pilocarpine solutions and found that browache occurred irrespective of concentration. Romano⁴⁵⁵ found conjunctival hyperemia, browache, and myopia more common with 2% aceclidine than with 2% pilocarpine. Contact dermatitis and urticaria are allergic reactions produced by topical pilocarpine.⁵¹⁵

Miosis and narrowing of the anterior chamber may occur less often with sustained-release membranes (Ocusert), but this difference tends to decrease with age.⁴³⁵ However, Drance and associates⁵¹⁶ found the decrease in visual acuity from induced myopia to be greater from Ocusert P20 than from 2% pilocarpine 90 minutes after placing the drop in one eye and the membrane in the other. They were reluctant to blame the difference in acuity purely on an accommodative cause because the spherical equivalents in the two eyes were similar. There was no interocular difference in acuity 90 minutes after placing a 4% pilocarpine drop in one eye and an Ocusert P40 membrane in the other. Worthen and co-workers⁴⁵⁸ reported that the membranes often were irritating and associated with browache, tearing, and discharge. Meyer⁵¹⁷ reported the development of acute glaucoma after the membrane was lost from an eye treated prophylactically with an Ocusert.

Poinoosawmy and colleagues⁵¹⁸ found that pilocarpine drops did not change human corneal thickness but did reduce the radii of the horizontal and vertical meridians of the cornea from 7.86 to 7.78 mm and from 7.90 to 7.81 mm, respectively. In addition to the corneal changes, there was a reduction in the radius of curvature of the anterior lens surface. In all 78 eyes in which pilocarpine produced a decrease in acuity, concave lenses were effective in returning the vision to predrop levels. Drance⁵¹⁹ found that pilocarpine and echothiophate altered scleral rigidity. In 58 human eyes, 68% had a decrease of less than 30% in scleral rigidity, 17% had a decrease of more than 30%, and 15% had an increase of more than 30%.

Kennedy and associates⁵²⁰ reported that patients receiving pilocarpine developed an atypical band keratopathy. However, it is believed that this was the result of the mercury-containing preservative, not the pilocarpine.^521–523 Mercury-containing preservatives are absorbed onto the rubber and plastic components of bottles and undergo degradation.^524,525

Miosis from pilocarpine drops significantly reduced the visual field area measured by Goldmann or automated techniques.^526,527 This is presumably due to reduced retinal illumination. Goldmann visual field area reductions for smaller isopters (I-2e = 64.5% ± 22.3%) were greater than for larger isopters (I-4e = 23.9% ± 16%) 30 minutes after normotensive subjects received 2% pilocarpine. In open-angle glaucoma subjects, a drop of 2% pilocarpine reduced automated visual field sensitivity by -1.49 dB.

Pilocarpine eyedrops increase the permeability of the blood-aqueous humor barrier.⁵²⁸ Normotensive subjects with darkly pigmented irides were given a single drop of 1% or 3% pilocarpine unilaterally and the vehicle contralaterally. Pilocarpine (1%) produced a significant increase in the mean ± SE anterior chamber protein content 3, 8, and 10 hours later. The increase at 8 hours, 21% ± 10%, was maximum. Pilocarpine (3%) produced significant elevations 1 to 4 and 6 hours postinstillation. The increase at 1 hour, 55% ± 11%, was maximum.

Chronic therapy of glaucoma with topical agents may alter the bulbar conjunctiva and reduce the success of subsequent filtering procedures. Rabbit eyes treated 4 months with pilocarpine, timolol, or saline had increased fibroblast proliferation after filtering procedures; epinephrine treatment did not produce this response. Pilocarpine produced the largest increase.⁵²⁹ One retrospective study could not confirm that long-term topical treatment of patients had a significant negative impact on subsequent filtering surgery.⁵³⁰ Another study was more suggestive of a deleterious effect but could not determine whether topical therapy in general, a specific drug class, or length of disease was the culprit.⁵³¹ In this study, 106 patients had conjuctival biopsies at the time of filtering surgery and were followed a minimum of 6 months. Specimens from those whose trabeculectomies failed had significantly more fibroblasts, macrophages, and lymphocytes. The surgical success rate in subjects treated less than 2 months, 86%, was not significantly different from that in subjects treated less than 3 years, 92%. However, patients treated with topical drugs for more than 3 years before surgery had a significantly lower surgical success rate, 30%.

The possibility of pilocarpine-induced lens opacities has been examined by several workers. de Roetth¹⁷⁹ reported anterior subcapsular vacuoles in 16% of eyes treated with 1% to 6% pilocarpine for a mean of 66 months and in 51% of eyes treated with 0.03% to 0.25% phospholine iodide for a mean of 20 months. In an age-matched group of normal eyes, 15% had vacuoles. In a retrospective study of patients treated for 3 years, Shaffer and Hetherington⁵³² found an 8% incidence of cataracts in an untreated control group, a 6% incidence in a group treated with pilocarpine, and a 38% incidence in a group treated with an anticholinesterase (DFP, demecarium, or echothiophate). Axelsson and Holmberg⁵³³ found about a fivefold increase in development and progression of cataracts in eyes treated with echothiophate compared with eyes treated with pilocarpine. These results do not support a cataractogenic role for pilocarpine.

Muscarinic agonists can produce a closed-angle glaucoma if the lens accommodates sufficiently to push the iris base against the trabecular meshwork^534–536 or sufficiently to block the miotic pupillary opening. Eyes with shallow chambers are more at risk, but occasionally the latter occurs in eyes with angles that were initially open.⁵³⁷

Specular and electron microscopic investigations of toxicity to rabbit endothelium from 1% acetylcholine and 0.01% carbachol (Miostat) failed to show permanent pathology.⁵³⁸ There were no temporary changes after in vitro corneas were bathed for 15 minutes with acetylcholine. After carbachol, changes in cell junctions were apparent within 1 hour but not thereafter. Intraocular 1% acetylcholine caused acute, reversible anterior subcapsular lens opacities in rabbits.⁵³⁹ Higher concentrations caused iritis in dogs.⁴⁷⁷ However, Rosen and Lazar⁵⁴⁰ pointed out that these effects probably were due to hyperosmolarity (e.g., the 1% acetylcholine solution was 390 mOsm). Similarly, Jay and MacDonald⁵⁴¹ found that intraocular injection of 1% pilocarpine or acetylcholine was not toxic to bovine endothelial cells unless the solutions were made significantly hyperosmolar or hypo-osmolar relative to aqueous humor (300 mOsm). Systemic absorption of acetylcholine after injection into the anterior chamber has, rarely, been accused of causing hypotension and bradycardia.⁵⁴² However, the inability of atropine to reverse the bradycardia makes it unlikely that acetylcholine absorption was at fault.

A cause-and-effect relationship between the topical use of muscarinic agonists and a resultant retinal detachment cannot be established because of the rarity of occurrence of the latter despite the frequency of use of the former; however, suspicions of causality remain. Both 1% pilocarpine ⁵⁴³ and membrane (Ocusert) pilocarpine delivery have been associated with retinal detachment.^544,545 A macular hole developed in an eye with a nondetached posterior vitreous face when pilocarpine therapy was initiated; the hole resolved on discontinuation of the drug.⁵⁴⁶

Indirect-Acting Muscarinic Agonists

GENERAL CONSIDERATIONS. The ocular effects of this class of drugs, the cholinesterase inhibitors, are qualitatively similar to those of the direct-acting agonists: miosis, ciliary muscle contraction, and ocular hypotension. The indirect agonists can be subdivided into anticholinesterases that carbamylate the enzyme and anticholinesterases that phosphorylate it. Physostigmine, neostigmine, pyridostigmine, and demecarium are examples of the former, and DFP and echothiophate are examples of the latter (Figs. 6 and 7). The carbamylating drugs are usually shorter acting and more acetylcholinesterase specific than the phosphorylating compounds (e.g., DFP is more active against serum cholinesterase [pseudocholinesterase, butyrylcholinesterase], whereas physostigmine is about equally active against acetylcholinesterase and serum cholinesterase). An exception is demecarium, which, although a carbamylator, is long acting.⁵⁴⁷ This is because it contains two carbamate groups; it can carbamylate at two sites and cause permanent structural alteration of the enzyme. Demecarium is somewhat more active against acetylcholinesterase than against serum cholinesterase.

There is evidence that multiple molecular forms of acetylcholinesterase exist and not all forms are equally susceptible to organophosphate inactivation.⁵⁴⁸ Topical application of organophosphates seems to produce complete inhibition of acetylcholinesterase in the anterior structures (e.g., the cornea and iris) but not the retina.⁵⁴⁹ The retina, probably because of its distance from the cornea, is partially protected. Recovery of acetylcholinesterase activity is characterized by more rapid synthesis of lower-molecular-weight forms of the enzyme, which in turn are assembled into the higher-molecular-weight forms.⁵⁵⁰

Echothiophate was first marketed in 1959, after Leopold and colleagues⁵⁵¹ had shown its clinical usefulness. DFP had been the phosphorylating anticholinesterase used previously, but it was unstable in water. One-tenth percent DFP in water was ineffective within 7 days.⁵⁵² The clinical efficacy of DFP in peanut oil was superior to that of DFP in petrolatum.⁵⁵³ In peanut oil, DFP remained active after 1 year of storage at room temperature and after 1 hour of autoclaving,⁵⁵⁴ and this was the usual method of dispensing it. Echothiophate, being a positively charged quaternary amine, penetrated the corneal epithelium less readily than DFP but had the advantage of much greater stability in water. After 5 weeks at room temperature, the solution retained 75% activity: at 10 weeks, there was 62% activity, and at 30 weeks, there was 55% activity.⁵⁵¹ Lawlor and Lee⁵⁵⁵ found that bottles of echothiophate solution refrigerated at 5°C for 8 weeks and opened daily lost only 4% to 5% of their activity. If stored at room temperature and opened daily, a 19% decrease in activity occurred in 8 weeks. Unopened solutions refrigerated 5 months retained 64% of their activity.

The phosphoryl group can be hydrolyzed off the esteradic site of cholinesterase by the oximes (RCH<cmb15>NOH). The oximes are much less effective in removing carbamyl-inactivating groups. By removing the phosphorylating group, the cholinesterase molecule is reactivated. However, if the phosphorylating agent becomes dealkylated while it is attached to the enzyme, that is, through hydrolytic loss of (CH₃)₂CH for DPF or CH₃CH₂ for echothiophate, then the drug cannot be removed from the enzyme. This process, called aging, results in a permanently inactivated cholinesterase molecule.⁵⁵⁶ An example of an oxime used to reactivate cholinesterase is pralidoxime (2-pyridine aldoxime methochloride, protopam, 2-PAM) (Fig. 8).

Like other oximes, pralidoxime can competitively bind to muscarinic receptors, but this is probably of no clinical significance.⁵⁵⁷ The drug is highly stable in solution; after storage at 37°C for 12 years, 75% of pralidoxime remained active.⁵⁵⁸

Pralidoxime is eliminated rapidly in the urine (80% to 90% of an orally administered dose is excreted in 60 to 90 minutes).⁵⁵⁹ Ocular penetration of pralidoxime after topical or systemic administration is limited because the molecule carries a positive charge at all times. Human volunteers given repeated oral and intramuscular doses of pralidoxime mesylate experienced diplopia and blurred vision.⁵⁶⁰ There was a 60% incidence of visual effects when subjects were given 4 g every 6 hours for 24 hours followed immediately by three 500-mg intramuscular injections, each injection being separated by 20 minutes. When rabbits were injected intramuscularly with pralidoxime mesylate 10 mg/kg, no drug was detected in the aqueous humor.⁵⁶¹ When the concentration was increased to 40 mg/kg, the drug was detectable in aqueous humor. Diacetylmonoxime (DAM) does not carry a positive charge and can readily penetrate the blood-eye and blood-brain barriers (see Fig. 8). However, it is not commercially available in the United States.

OCULAR PHARMACOLOGY. Iris Sphincter Muscle. The carbamylating and phosphorylating anticholinesterases have no intrinsic cholinergic agonist activity. They act only by preventing acetylcholine hydrolysis. In cats with ciliary ganglionectomies, DFP did not produce miosis, nor did a 10% acetylcholine drop have significant effect.⁵⁵² However, after DFP application, a 1% acetylcholine eyedrop produced miosis for 9 to 13 days.

Because cholinesterase inhibitors act through the accumulation of acetylcholine at the muscarinic receptor, drugs that compete with acetylcholine can alter the response to these indirect agonists. Pilocarpine has less intrinsic muscarinic activity than acetylcholine. Thus, when maximally effective doses of physostigmine or DFP are given, the subsequent addition of pilocarpine causes a diminution of both miosis and hypotension.³⁶⁷ Kaufman and Barany^562,563 found similar results in primate eyes pretreated with cholinesterase inhibitors. However, if the inhibition of acetylcholinesterase is sufficiently submaximal (e.g., as when produced by low concentrations of physostigmine), then additional pupillary constriction can be achieved by giving pilocarpine drops.³⁸⁰ The mydriasis and cycloplegia from 3 drops of 1% atropine, given within a 36-hour period, are partially reversed by 2 to 3 drops of 0.5% echothiophate, given within a 30-minute period.⁵⁵¹ Single instillations of 0.5% demecarium overcome mydriasis from either 1% atropine or 4% homatropine; within 1 to 3 hours of demecarium administration, the pupil is smaller than normal. This miosis becomes maximum at 24 hours and requires up to 7 days to recover. However, if the 0.5% demecarium were given first and the atropine or homatropine were instilled 6 hours later, the initial miosis would be abolished within 10 minutes.

Bito and Banks³⁷³ observed that monkeys given 0.1% DFP drops twice a day developed maximum miosis on the second day of treatment, but by the sixth day of treatment, the pupils had returned to pretreatment diameters in both dark and light. Increased iris dilator tone was not producing this phenomenon because it occurred despite prior superior cervical ganglionectomy.⁵⁶⁴ Subsensitivity of the muscarinic receptors may have been the cause, or a toxic effect of DFP on the iris sphincter may have been at fault.⁵⁶⁵ Soli and associates⁵⁶⁶ reported a similar finding in guinea pigs. Single drops of cholinesterase inhibitor produced marked miosis, but by the third day of daily treatment, a decreased response was found. They could not explain this effect either by a reduced number of muscarinic receptors, using quinuclidinyl benzilate binding, or by a more rapid synthesis of acetylcholinesterase. Furthermore, the miotic response to light stimulation remained, throwing into question the theory that drug toxicity of iris musculature was at fault. The rat iris dilator muscle has been shown to relax when exposed to low acetylcholine concentrations but to constrict, producing mydriasis, at 1 μmol/L and higher concentrations.⁵⁶⁷ Perhaps this would explain the decreased miosis. Another theory relates to choline uptake. Both carbamylating and phosphorylating cholinesterase inhibitors reduce rat iris choline uptake.⁵⁶⁸ Such a reduction could eventually impair acetylcholine synthesis. However, administration of an exogenous agonist should result in miosis. When Bito and Baroody⁵⁶⁹ topically applied pilocarpine to monkey eyes made subsensitive by daily cholinesterase inhibitor drops, they found a reduced miotic response in echothiophate-treated eyes and no miotic response in DFP-treated eyes.

Dunphy⁵⁷¹ provided evidence that acetylcholine is continuously being released by ciliary ganglion neurons even in the absence of light stimulation or the near synkinesis. A volunteer had a single drop of 0.05% DFP instilled. He was kept in the dark, yet miosis occurred. Mindel⁵⁷⁰ demonstrated spontaneous acetylcholine release by denervated ciliary ganglia. Patients whose oculomotor nerves had been severed intracranially responded to an eyedrop of cholinesterase inhibitor with a marked miosis. Presumably, the amount of spontaneous acetylcholine released was insufficient to stimulate the iris sphincter until the cholinesterase inhibitor allowed the transmitter to accumulate. Loewenfeld and Newsome⁵⁷² found that 8 minutes after a drop of 0.5% physostigmine, the increased effectiveness of released acetylcholine allowed the iris to react more rapidly and completely to light stimulation.

In normal subjects, miosis begins 5 to 10 minutes after instillation of 0.1% DFP, 10 to 45 minutes after instillation of 0.5% echothiophate, and 45 to 60 minutes after instillation of 0.1% to 0.5% demecarium. Maximum miosis occurs within 20 minutes of DFP application, whereas miosis from demecarium requires 2 to 4 hours. Concentrations of DFP below 0.01% have no effect. Recovery from the miosis of DFP, echothiophate, or demecarium requires 3 to 28 days.^547,551,552

If physostigmine were given 15 minutes before DFP, the miotic effect would be that of the former (i.e., recovery requires only 2 to 3 days).⁵⁵³ This demonstrates that both carbamylating and phosphorylating cholinesterase inhibitors attack the same site on acetylcholinesterase and in a noncompetitive manner. The first drug to reach the esteradic site will inactivate it until spontaneous hydrolysis removes the carbamyl or phosphoryl group.

Ciliary Muscle. Strips of ciliary muscle from cats, chickens, frogs, rabbits, and monkeys exposed to acetylcholine had their contractions increased by factors of 10 to 10,000 if physostigmine 1 μg/mL was in the bathing solution.⁵⁷³ Atropine was effective in relaxing muscle strips that had previously received physostigmine.

Monkeys treated for 5 to 6.5 months with topical echothiophate had approximately a 65% reduction in their ciliary muscle muscarinic binding sites and a decreased response to pilocarpine. The binding affinity of the remaining muscarinic receptors was unchanged.^574,575 The iris response to pilocarpine was unaffected. After echothiophate treatment was discontinued, the number of ciliary muscle muscarinic binding sites and the response to pilocarpine returned in 4 to 8 weeks. Eyes permitted to recover for 5 to 6.5 months exhibited a rebound effect (i.e., their ciliary bodies had more than double the number of muscarinic bindings sites compared with untreated control monkeys). When 0.05% physostigmine was applied topically to human subjects, an accommodation of the lens could be elicited that was above physiologic levels.⁵⁷⁶ In three subjects, aged 27 to 30 years, this increase was more than 2 D. Physostigmine (0.05%), by facilitating accommodation, also produced a decrease in the accommodative convergence/accommodation (AC/A) ratio (e.g., in one subject the AC/A ratio was 1.2 to 1.3 before physostigmine and 0.8 to 0.9 after physostigmine). Wilkie and co-workers³⁹⁴ measured human anterior chamber depth in response to echothiophate. A single drop produced little shallowing, but there was a progressive decrease in depth during the 10-week treatment period.

Intraocular Pressure. DFP causes an initial rise in rabbit intraocular pressure.^577,578 The pressure elevation begins with the onset of miosis and at its maximum, approximately 30 minutes after instillation, is 15 to 30 mmHg above baseline. The pressure remains above baseline 4 to 6 hours later. Intravenously administered fluorescein accumulates in the anterior chamber, indicating that the blood-aqueous barrier has been impaired.⁵⁷⁹ Histologic examination shows epithelial blisters on the ciliary processes, engorgement of the iris and ciliary vessels, and increased protein in the anterior and posterior chambers.⁵⁷⁷ Prior phenylephrine, injected subconjunctivally, prevents the elevations in anterior chamber protein and intraocular pressure, presumably by constricting the uveal vessels.⁵⁷⁸

Monkey eyes treated for 5 or more months with topical echothiophate drops had elevated intraocular pressures associated with histologic evidence of damage to the aqueous humor outflow system.⁵⁸⁰ Extracellular material was found in the trabecular meshwork, Schlemm's canal was altered in shape, and there was discontinuity between ciliary muscle bundles and trabecular beams.

McDonald⁵⁸¹ reported that 10% of human eyes treated with DFP also had an initial rise in intraocular pressure of 3 to 7 mmHg. Krishna and Leopold⁵⁴⁷ and Drance and Carr⁵⁸² found similar rises after demecarium and echothiophate administration. Some of the theories offered to explain these increases were that congestion of the ciliary body vessels caused a narrowed filtration angle, that increased protein in the aqueous humor drew water into the anterior chamber by osmosis, and that ciliary muscle contraction caused a forward shift of the lens-iris diaphragm.⁵⁸³

The eventual hypotensive effect of cholinesterase inhibitors is variable in normotensive eyes. Leopold and colleagues⁵⁵¹ found it to be rarely more than 2 to 3 mm. Drance and Carr⁵⁸² reported a 52% decrease in pressure. In most normotensive subjects the intraocular pressure after a single instillation begins to decline in approximately 30 minutes without transient pressure rise. Although miosis begins about the same time as the hypotensive action, the two do not always occur together.⁵⁸² With 0.1% to 0.5% demecarium administration, the maximum hypotensive effect occurs 24 hours after instillation and may remain below baseline for as long as 9 days. A significant increase in tonographic outflow facility is usually present 2 hours after instillation and is almost always present 24 hours after instillation.^547,584 However, the hypotensive action is not always associated with an increase in outflow facility.⁵⁸⁵ Drance⁵⁸⁵ found that instillation of single drops of 0.1% or 0.25% demecarium in normotensive eyes did not produce hypotensive effects until after latency periods of 12 hours. The tonographic increases in outflow facility lasted up to 5 days. However, in four eyes there were no increases in outflow facility despite large decreases in intraocular pressure.

Retina. Infusion of physostigmine into the rabbit retinal circulation abolished the directional specificity of both on-center and on-off retinal ganglion cells.⁵⁸⁶

Extraocular Muscles. Topical application of DFP to rabbit eyes resulted in increased sensitivity of the superior recti muscles to solutions of acetylcholine and carbachol (i.e., the muscles produced larger contractions than in control eyes).⁵⁸⁷ Recovery was gradual. More than 7 days without DFP treatment was needed for sensitivity to return to normal. DFP is highly lipid soluble. It was believed that DFP penetrated the conjunctiva and inhibited acetylcholinesterase in the neuromuscular junctions.

THERAPEUTICS. Glaucoma. Physostigmine in concentrations of 0.25% to 1% increases outflow facility and lowers intraocular pressure in subjects with initial readings of 22 mmHg or greater.⁴²³

Demecarium is a potent hypotensive agent.^588,589 Drance⁵⁸⁵ used demecarium to treat 40 eyes with chronic simple glaucoma. In 38, there was a decrease in intraocular pressure. The mean time for maximal decrease in pressure was 34 hours, and the mean percent fall in pressure for all 40 eyes was 48%. In 34 eyes tested by tonography, the mean increase in outflow facility was 121%.

Phosphorylating agents are also effective. DFP, used by Leopold and McDonald⁵⁵³ over a period of 4 or more months, lowered the ocular pressures of 71% of eyes with chronic narrow-angle glaucoma, 74% of eyes with aphakic glaucoma, and 54% of eyes with open-angle glaucoma. It was of value in controlling 61% of eyes with uveitic (inflammatory) glaucoma and in terminating acute glaucoma attacks. Only 14% of acute attacks were terminated by the use of pilocarpine, acetyl β-methylcholine, physostigmine, or neostigmine, whereas 57% ceased after the application of DFP. However, Leopold and McDonald⁵⁵³ also noted six glaucoma patients in whom DFP produced an unexpected increase in intraocular pressure. Four had preexisting narrow angles. The maximum effective concentration of DFP in peanut oil was 0.2%. These authors found that pilocarpine and/or physostigmine was as effective as 0.1% DFP in lowering ocular pressures below 30 mmHg in 17 glaucoma subjects. However, in 29 other subjects, DFP was successful when the other two were not.

Echothiophate has been the most widely used phosphorylating cholinesterase inhibitor. In 59 chronic simple glaucoma patients given a single drop of 0.1% to 0.25% echothiophate, the mean decrease in ocular pressure was 48% 3 hours postinstillation, and the mean increase in outflow facility, measured in 28 eyes 24 hours after instillation, was 127%.⁵⁸² Echothiophate, unlike pilocarpine and carbachol, produced a sustained increase in outflow facility.⁴⁰⁶ In dilutions as low as 0.01%, given twice a day, it was as effective as pilocarpine in 42% of patients.⁵⁹² Harris⁵⁹³ studied 15 glaucoma patients and found that maximum improvement in intraocular pressure and outflow facility occurred with 0.06% echothiophate; increasing the concentration to 0.25% did not significantly improve either (Table 1). The maximum effect on intraocular pressure and outflow facility has been reported to occur from 4 to 27 hours after application.^406,594

TABLE 1. Results of Studies of Echothiophate in 15 Glaucoma Patients

There is evidence to support Barsam's⁴⁰⁶ belief that echothiophate and pilocarpine are similar in hypotensive potency and differ only in duration of action. In patients on chronic therapy, the minimum intraocular pressure on the diurnal curve is a measure of drug potency, and the magnitude of the fluctuation of the diurnal curve is a measure of drug duration of action. Drance⁵⁹⁴ had compared the diurnal hypotensive efficacy of 4% pilocarpine four times a day with that of 0.06% echothiophate two times a day using subjects with ocular hypertension and chronic simple glaucoma. Seventy percent of pilocarpine-treated eyes had diurnal pressure peaks that rose above 24 mmHg as opposed to only 20% of echothiophate-treated eyes. Similar results were reported by Barsam and Vogel,⁵⁹⁵ comparing 0.06% echothiophate once a day with 2% pilocarpine four times a day. Pratt-Johnson and colleagues⁴⁵¹ studied the pressure response to 0.06% echothiophate twice a day and to 4% pilocarpine four times a day in 20 eyes with intraocular pressures greater than 24 mmHg. The mean peak pressure while receiving pilocarpine, 23 ± 5.3 mmHg, was greater than that while receiving echothiophate, 19 ± 5.5 mmHg. However, the mean minimum pressure achieved with pilocarpine, 13 ± 2.7 mmHg, was similar to that achieved with echothiophate, 12 ± 2.8 mmHg. Either the two drugs are equally potent or the 0.06% echothiophate solution is too dilute to evoke a maximum hypotensive effect.

When demecarium or echothiophate was added to the various therapies used by poorly controlled glaucoma patients, the results indicated that the two drugs were about equally effective.⁵⁹⁶ After 6 months of therapy, 0.25% and 1% demecarium maintained 27% and 36% of eyes, respectively, at a pressure less than 20 mmHg, and 13% and 20%, respectively, had an outflow facility of more than 0.15 μL/min/mmHg. Using these same criteria, 0.25% echothiophate controlled the pressures in 25% of eyes and improved the outflow values in 20% of eyes. Of 42 eyes with pressures uncontrolled on echothiophate therapy and 32 eyes with pressures uncontrolled on demecarium therapy, demecarium was effective in 13% of the former and echothiophate was effective in 24% of the latter.

Echothiophate has been used in combination with epinephrine, acetazolamide, and pilocarpine.^555,597 Klayman and Taffet⁵⁹⁸ found that 0.06% echothiophate, twice a day, controlled for at least 1 year the intraocular pressure of 43% of eyes with pressures greater than 20 mmHg and visual field loss. Another 33% required the addition of acetazolamide or epinephrine to bring the ocular pressures below 21 mmHg. In 7% of eyes the pressures were controlled when all three drugs were used. The pressures in 17% of eyes remained uncontrolled despite therapy with all three drugs.

Accommodative Esotropia. Atropine was occasionally used in the past to treat accommodative esotropia. With the onset of cycloplegia, the esotropia would increase as the patient attempted to focus. Then, as this inability was accepted, accommodative efforts would cease and the eyes would straighten. To varying degrees, however, near work would once again elicit volitional accommodative effort and the eyes would deviate inward. In contrast to atropine, cholinesterase inhibitors correct accommodative esotropia by facilitating peripheral accommodation (i.e., they decrease the need for volitional accommodative effort). Although they also induce miosis and thereby increase the depth of focus, this is not their primary mechanism of action.⁵⁹⁹ If the AC/A ratio is abnormally high, cholinesterase inhibitors will lower it. Cholinesterase inhibitors would then be expected not only to facilitate accommodation but also to produce an accommodative myopia in nonpresbyopic patients. However, there is some evidence that cholinesterase inhibitors are less likely to produce a spasm of accommodation than are the direct-acting agonists, such as pilocarpine.^600,601 Rehany and associates⁶⁰² found that a single drop of 0.125% echothiophate failed to induce accommodation during the 24 hours after instillation, whereas a single drop of 4% pilocarpine produced a demonstrable accommodation that lasted several hours, being more than 4 D at its peak 30 minutes after instillation. Miller⁶⁰⁰ found that patients treated with DFP, demecarium, and echothiophate had less than 0.5 D of induced accommodation after 1 week of therapy.

Wheeler⁶⁰³ used cholinesterase inhibitors to diagnose accommodative esotropia, before prescribing spectacles, and to treat the condition. Both carbamylating agents (e.g., 0.25% physostigmine) and phosphorylating agents (e.g., 0.1% DFP) were effective.⁴⁷⁴ However, the latter were longer acting. Miller found 0.025% DFP,⁶⁰⁰ 0.25% demecarium, and 0.1% echothiophate about equal in controlling accommodative esotropia.

Parks⁶⁰⁴ used a tapering dose of anticholinesterase to wean children off therapy over a 6-week to 18-month period. As fusional capabilities gradually increased, less medication was required. Lasting improvement occurred in 27% of patients under 5 years of age and in 88% of children 7 to 12 years of age. The overall success rate was 68%. Parks concluded that cholinesterase inhibitors were more likely to give permanent improvement if they were discontinued after age 7 rather than before. Children who develop accommodative esotropia within the first year of life form a special subgroup. Approximately 50% of these patients develop a nonaccommodative esotropia requiring surgery, even though they initially respond to echothiophate or DFP treatment.⁶⁰⁵

Synechiae. Cholinesterase inhibitors have been reported to occasionally break posterior synechiae that could not be lysed by dilatation. The rationale for their success was that the iris sphincter muscle provided a more forceful contraction than the iris radial muscle. Mamo and Leopold⁶⁰⁶ initially used anticholinesterases on two subjects with long-standing synechiae; they were unimpressed by the results. Byron and Posner⁶⁰⁷ reported 10 patients with posterior synechiae to the lens capsule and nine patients with posterior synechiae to secondary membranes. These subjects were initially made miotic with two drops of echothiophate. The echothiophate effect was then reversed by injecting 0.1 to 0.2 mL of 5% pralidoxime subconjunctivally. Maximum mydriasis was then achieved with two drops each of 1% atropine and 10% phenylephrine. Dense synechiae did not respond. There was partial to total lysis of smaller synechiae in 8 of the 10 patients with adhesions to the lens capsule. In 5 of 9 patients with adhesions to secondary membranes, there were increases of at least 2 mm in the pupillary diameters.

Cyclodialysis Clefts. Gonioscopic examination has verified that surgically created cyclodialysis clefts remained open postoperatively when these eyes were treated with cholinesterase inhibitors. Gorin⁶⁰⁸ found that a drop of 0.1% DFP daily for several weeks was effective in 15 patients.

Refractive Errors. Cholinesterase inhibitors have been used instead of spectacles to correct myopia, presbyopia, and hyperopia.⁶⁰⁹ Their effectiveness in the first two conditions was attributed to the marked miosis, producing a pinhole effect. Echothiophate (0.06%), twice a day, was the initial dose, with the frequency and concentration increased as needed. In presbyopes, the criterion for success was maintenance of best distance acuity with improvement of near reading acuity to Jaeger 3; in hyperopes, the criterion was 20/25 (6/7.5) distance vision. Fifty-five percent of presbyopes no longer required spectacles and 26% required only a distance prescription. In hyperopes and myopes in the + 2.00 D to -2.00 D range there was a 42% success rate. In patients with errors greater than this, there was a 12% success rate.

Adie's Syndrome. Variable success has been reported with dilute concentrations of physostigmine to reduce the mydriasis and improve the accommodation in eyes with Adie's syndrome.⁶¹⁰ Eyedrops of 0.0125% to 0.125% physostigmine were given to five patients. Two patients tolerated long-term treatment, but three patients discontinued their use because of symptoms of miosis, blur, or discomfort.

OCULAR TOXICITY AND SIDE EFFECTS. Since the mid 1960s, there has been considerable emphasis on the toxicities and side effects of the cholinesterase inhibitors.

Cataractogenesis. Harrison⁶¹¹ is credited with first reporting an organophosphate-induced lens opacity. The patient was a 13-year-old girl given 0.025% DFP in peanut oil nightly for 3 months. Anterior subcapsular lens opacities were noted. The medication was stopped and in 9 days the opacifications had partially cleared. In 3 weeks, the lenses appeared normal. Pietsch and co-workers⁶¹² found no lens changes in children treated up to 48 months with echothiophate. Although lens opacities rarely occur in children, this is not true of adults. Axelsson and Holmberg⁵³³ found that in glaucoma patients treated for a mean of 22 months with 0.06% or 0.25% echothiophate, new opacities developed at a rate more than six times greater than that in glaucoma patients treated for the same period with 2% or 4% pilocarpine. Opacities developed in 40% of the echothiophate-treated patients but in only 6% of the pilocarpine-treated patients. Progressive pathologic changes also occurred in subjects with preexisting opacities. About five times as many patients on echothiophate progressed compared with those on pilocarpine. A positive correlation existed for strength as well as for length of treatment. DFP and demecarium were also cataractogenic.^532,613 In monkeys, carbachol, which has some cholinesterase inhibitory action, has produced cataracts.⁶¹⁴ In a prospective study of 16 patients observed for 7 to 21 months, there were no lens changes in those 6 subjects receiving 0.06% or 0.125% echothiophate.⁶¹⁵ However, 5 of 10 subjects receiving 0.25% echothiophate developed anterior subcapsular vacuoles, nuclear sclerosis, and/or posterior subcapsular cataracts. Some authors have stated that anterior subcapsular changes occurred earliest, beginning with vacuoles and progressing to a mossy-appearing opacification.⁵³² Axelsson⁶¹⁶ and Morton and colleagues⁶¹⁷ noted that when echothiophate therapy was discontinued, the lens changes progressed in some eyes whereas in others they regressed. Occasionally, the lens changes regressed despite continued treatment.

In monkeys, concomitant treatment with atropine seemed to delay the onset of lens changes and in some instances seemed to partially reverse lens changes produced by echothiophate.⁶¹⁸ Levene⁶¹⁹ noted that in 10 of 12 patients developing echothiophate-related cataracts, pilocarpine had been used as prior therapy for 2 months or less. He suggested that 3 months or more of pilocarpine pretreatment might protect against lens changes. However, many subjects with cholinesterase inhibitor-induced cataracts had received prior long-term pilocarpine therapy. In Shaffer and Hetherington's⁵³² study, all their subjects received prior pilocarpine therapy, but DFP, demecarium, and echothiophate use was associated with 30%, 40%, and 35% cataract incidences, respectively. Axelsson⁶¹⁶ found that there was a 50% incidence of decreased vision in eyes treated initially and solely with echothiophate; in eyes previously treated with pilocarpine or pilocarpine-physostigmine, the incidence was about the same (43%).

The biochemical abnormality responsible for the development of cataracts has not been defined. The phosphorylating agents are highly reactive and may attack multiple enzymes and cell structures. Harkonen and Tarkkanen⁶²⁰ gave albino rabbits 0.25% echothiophate eyedrops twice daily for 15 weeks. No lens opacities occurred, but ATP content decreased by 35% and lactate content by 17%.

Iris Nodules. Abraham⁶²¹ reported that two thirds of 66 young subjects treated with DFP for strabismus developed iris nodules. Only one subject in the study was over age 14 years. Nodules appeared after a mean of 10 weeks of treatment. A nasal pupil location was favored. In all cases, the nodules disappeared after discontinuation of treatment, leaving shrunken tags. In 83%, the nodules were gone within 15 weeks; by 42 weeks, all had shrunken. The tags usually, but not always, disappeared gradually. Concentrations as low as 0.03% echothiophate have been reported to produce nodules.⁵⁹² Therapy with pilocarpine and physostigmine also led to nodule formation, but much less often. Microscopic examination revealed hyperplasia of the iris pigment epithelium, forming solid elevations more often than cysts.⁶²² Phenylephrine (10%) given nightly appeared to prevent the development of these nodules.⁶⁰³

Retinal Detachment. Because the human retina stretches during marked ciliary muscle contraction (e.g., 9 D of accommodation can cause 4% to 5% retinal stretching),⁶²³ it is not surprising that retinal tears have been associated with cholinesterase inhibitor use. However, the incidence is too low to allow a statistical analysis of causality. Instead, the literature on this subject consists of case reports.^596,624 Physostigmine, the first drug used to treat glaucoma, was reported to be clinically effective by Leber in 1876. In 1877, he reported a retinal detachment associated with therapy. Pape and Forbes⁴⁹¹ and Alpar⁶²⁵ reported detachments in association with direct-acting muscarinic agonists, such as pilocarpine, as well as with indirect agonists. In the former paper, 41% of detachments occurred within 2 months of the initiation of therapy, but the range extended to 17 years.

Acute Closed-Angle Glaucoma. Leopold and McDonald⁵⁵³ found DFP to be of value in treating narrow-angle glaucoma. However, the cholinesterase inhibitors subsequently have been shown to provoke attacks in predisposed eyes.^551,583

Miscellaneous. Headache, browache, eyeache, induced myopia, conjunctival congestion, allergy, punctal stenosis, fibrinous iritis, and posterior synechiae have been associated with the use of cholinesterase inhibitors.^{547,553,596,626} In over 70% of subjects with side effects, the signs and symptoms appeared within the first 48 hours of treatment. The aching usually did not persist beyond the first week of treatment and was often relieved by salicylates. Side effects caused 14% of demecarium-treated patients and 20% of echothiophate-treated patients to discontinue therapy. Approximately 25% of the former and 50% of the latter were successfully changed to the other drug. Using lower drug concentrations appeared to reduce the frequency of side effects, but all the same problems still occurred.^551,592,593

SYSTEMIC TOXICITY AND SIDE EFFECTS. Leopold and Comroe⁵⁵² reported that 0.1% DFP in peanut oil was absorbed sufficiently after topical eyedrop use to lower serum pseudocholinesterase activity but not red blood cell acetylcholinesterase activity. Pilocarpine therapy, however, was not associated with a reduction in serum or red blood cell cholinesterase activity.⁶²⁷ In glaucoma patients treated with echothiophate twice a day, the 0.03% and 0.06% concentrations reduced serum cholinesterase activity by 35% to 40% and red blood cell acetylcholinesterase activity by 25% to 30% within 2 weeks⁶²⁸; this level of reduction was maintained while therapy was continued for the ensuing year. Twice-a-day application of the more concentrated echothiophate solution, 0.25%, resulted in a 75% to 80% reduction in serum cholinesterase and an 80% to 85% reduction in red blood cell acetylcholinesterase activity. These, too, occurred within 2 weeks. Upon cessation of therapy, cholinesterase activity began to increase within a week. Recovery was complete by 4 months.⁶²⁹ Humphreys and Holmes⁶³⁰ and Wahl and Tyner⁶³¹ believed that systemic side effects, such as diarrhea⁶³² and nausea, were inversely correlated with blood cholinesterase activity; however, de Roetth and co-workers⁶²⁸ did not (e.g., one of their subjects who was symptom free had only 5% of pretreatment blood cholinesterase activity).

The pupils are usually miotic in patients with systemic anticholinesterase toxicity unassociated with ocular use (e.g., insecticide poisoning). However, Dixon⁶³³ reported two patients with organophosphorus toxicity who had dilated pupils. Nattel and colleagues⁶³⁴ studied the effect of intravenous physostigmine, 2 mg, on the pupils of patients with drug-induced coma. In subjects comatose from drugs without atropinic activity (e.g., barbiturates), physostigmine caused pupils to dilate. In subjects comatose from drugs with atropinic activity (e.g., amitriptyline), physostigmine did not cause pupil dilatation. These results are difficult to interpret and may have been due to central nervous system effects. Physostigmine, being a tertiary amine, can cross the blood-brain barrier.

Phosphorylating inhibitors depress serum cholinesterase activity and prolong the effects and increase the toxicities of drugs normally inactivated by this enzyme. Succinyldicholine and ester local anesthetics such as procaine are commonly cited in this regard. However, it is difficult to find instances in which chronic administration of eyedrops has depressed serum cholinesterase activity to a clinically significant degree. With regard to the prolongation of succinyldicholine effect, the report frequently referred to is that by Pantuck.⁶³⁵ It consists of a single case. A 39-kg female, on daily 0.125% echothiophate drops bilaterally for 9 months, was given 40 mg succinyldicholine intravenously. Diaphragmatic activity and tracheal tug did not begin for 10 minutes, but by 20 minutes postinjection her respirations had returned to normal. Serum cholinesterase activity was found to be 30% of normal levels. This relatively mild prolongation of apnea does not seem to justify the degree of fear that is often voiced. With regard to local anesthetic toxicity, there is even less justification. Again, a firm theoretic and experimental basis exists,⁶³⁶ but the amount of cholinesterase depression from systemic absorption of eyedrops seems inadequate to convert a therapeutic dose into a lethal one.^637,638 Other drugs shown to be hydrolyzed by serum cholinesterase are methylprednisolone acetate,⁶³⁹ substance P,⁶⁴⁰ and heroin.⁶⁴¹ The evidence that maternal cholinesterase-inhibitor eyedrops reduce fetal serum cholinesterase activity is limited.⁶⁴²

The human blood complement system, consisting of 20 plasma proteins and 9 regulatory proteins, neutralizes and lyses bacteria and viruses. Activation of this system usually requires two serine esterases, C1_R and C1_S, and an esteradic site on C2. The phosphorylating cholinesterase inhibitors, such as DFP, are potent inhibitors of these serine esterases.⁶⁴³ There are no reports of increased susceptibility to infections in patients receiving topical cholinesterase inhibitors.

ANTIDOTES. A small dose of intravenous physostigmine protects against a subsequent lethal dose of DFP. It also prevents a prolonged decrease in serum cholinesterase activity.⁶⁴⁴ However, physostigmine does not protect or reactivate serum esterase if it is given after DFP. Oxime therapy will reactivate serum cholinesterase if it is given soon enough. After 9 months of 0.25% echothiophate eyedrops twice a day, 1 week of pralidoxime, 500 mg orally three times a day, did not elevate serum or red blood cell cholinesterase activity.⁶²⁹ This was because the phosphorylated enzymes had aged. After 3 weeks of oxime treatment there was a significant increase in red cell acetylcholinesterase activity.⁶⁴⁵ This resulted from erythrocyte turnover and synthesis of new enzyme. In chronic simple glaucoma patients receiving 10.5 g pralidoxime orally each day for 3 weeks, there were no changes in pupil size, intraocular pressure, or outflow facility.⁶⁴⁵ de Roetth and associates⁶²⁹ reported that the intraocular pressures rose in patients given oral pralidoxime, but these subjects had discontinued their echothiophate eyedrops.

Local ocular oxime therapy has been investigated. Pralidoxime, diacetyl monoxime, TMB-4 (1,1-trimethylene bis-4-formylpyridinium oxime bromide), and MINA (monoisonitrosoacetone) have been instilled as 1%, 3%, and 5% aqueous drops, 5% aqueous drops with 1/3000 benzalkonium, and 5%, 10%, and 20% petrolatum ointments.⁶⁰⁶ In 31 volunteers, they had no effect on the normal pupil or in pupils made miotic with pilocarpine. With a few exceptions, multiple drops, even with benzalkonium, had no effect when given immediately after DFP- or physostigmine-induced miosis. The 10% and 20% ointments were effective antidotes in about half the subjects. Subconjunctival injection of a 5% solution was 100% effective in reversing miosis and was more effective than 1% atropine plus 10% phenylephrine eyedrops.⁶⁰⁷ Similar results were reported by Becker and associates⁵⁹⁷ in glaucomatous eyes. In untreated glaucoma patients, subconjunctival pralidoxime had no effect. However, in echothiophate-treated patients and, to the authors' surprise, in two of six pilocarpine-treated eyes, it produced a decrease in outflow facility.

Muscarinic Antagonists

GENERAL CONSIDERATIONS. The muscarinic blocking agents in clinical use act by competing with the physiologic agonist, acetylcholine, for the receptor. This may be too simplistic a view, however. Marshall⁶⁴⁶ studied the two stereoisomers of atropine: (-) hyoscyamine was 30 times more active than (+ ) hyoscyamine; (-) hyoscyamine was a competitive antagonist, but (+ ) hyoscyamine appeared to be a noncompetitive antagonist. Gupta and co-workers⁶⁴⁷ studied the binding of an irreversible antagonist, benzilylcholine mustard, to the muscarinic receptor. They found evidence that binding occurred at two different sites. Ellenbroek and colleagues⁶⁴⁸ believed that atropinic drugs were competitive antagonists but that agonists and antagonists bound to the receptor at different sites.

The muscarinic antagonists can be subdivided into the naturally occurring agents, such as atropine (hyoscyamine) and scopolamine (hyoscine), and the synthetic agents, such as cyclopentolate, homatropine, and tropicamide (Figs. 9 and 10). All are quite stable.

When cyclopentolate spontaneously degrades, it is by hydrolysis of its ester bond with formation of N,N-dimethylaminoethanol and cyclopentanyl benzeneacetic acid. The latter then either breaks down into phenylacetic acid and cyclopentanone or is hydroxylated and becomes hydroxycyclopentyl benzeneacetic acid.⁶⁴⁹ Atropine spontaneous hydrolysis is pH and temperature dependent. At 20° C, the half-life of atropine in a pH 7 solution is 2.7 years; in a pH 6 solution, 27 years; in a pH 5 solution, 266 years; and in a pH 4.5 solution, 811 years. At 30° C, these values are reduced to 0.61 years at pH 7, 6.1 years at pH 6, 61 years at pH 5, and 194 years at pH 4.5.⁶⁵⁰ The half-life of tropicamide is even longer (e.g., at 20° C and pH 7.45, it is 2346 years).⁶⁵¹ Additional factors, besides pH and temperature, are important for commercial preparations. Unbuffered solutions of atropine and scopolamine are more stable than buffered solutions, and low-density polyethylene containers are preferable to glass.⁶⁵²

Tropicamide, with pKa = 5.37, is the only clinically used muscarinic antagonist that is primarily (approximately 98%) in the nonionized form at physiologic pH. Homatropine, with pKa = 9.9, is only 0.32% nonionized. Atropine, pKa = 9.8, and cyclopentolate, pKa = 8.4, are also primarily in an ionized state.^653,654 As a result, tropicamide has much greater access to the muscarinic receptors because it can readily penetrate the corneal epithelium. The relative in vitro muscarinic blocking activities of these drugs is atropine > cyclopentolate > homatropine > tropicamide. However, the greater penetration of tropicamide makes it more effective than homatropine and changes the in vivo order of potency to atropine > cyclopentolate > tropicamide > homatropine.⁶⁵⁴ Tropicamide consists of two stereoisomers.⁶⁵⁵ Both appear to be competitive blockers of acetylcholine.⁶⁵⁶ However, the levorotatory isomer is far more active pharmacologically. In pigmented and nonpigmented irides, it is 75 times and 50 times, respectively, more active than the dextrorotatory isomer.

Muscarinic receptors in different tissues, and even in different mammalian species, tend to bind the non-subtype-specific antagonist atropine in a similar manner. The concentration of atropine needed to half-saturate human iris receptors in vitro is 0.4 to 0.7 nmol/L.⁶⁵⁷ This value agrees with those reported for rabbit iris,⁶⁵⁴ guinea pig ileum,⁶⁵⁸ and guinea pig heart.⁶⁵⁹ However, the variability in duration of action of muscarinic antagonists at different sites may be due to differences in receptor subtype (e.g., the tachycardia effect of atropine lasts minutes to hours, but the pupil dilation effect lasts for days). Two other factors that may affect duration of action are the pigment binding of drugs and, in some species, the presence of enzymes that inactivate muscarinic antagonists. Chen and Poth⁶⁶⁰ noted that eucatropine was a more effective mydriatic in white subjects than in blacks; the effect on Orientals was intermediate. Patil and associates⁶⁶¹ found that the binding to uveal pigment was not stereoselective. Salazar and co-workers^662,663 found that pigmented rabbit iris accumulated much more atropine than nonpigmented iris. On repeated washing, the atropine bound to nonpigmented tissues was removed more readily. Pigmented human iris tissue behaved in a manner similar to that of pigmented rabbit iris tissue. Pigment binding explains several clinical phenomena. There is a delay in onset and a decrease in magnitude of dilation in pigmented subjects. These result from an initial competition for atropine between pigment and receptors. There is a prolonged effect from atropine in pigmented subjects. It results from the melanin-bound drug acting as a reservoir. Emiru⁶⁶⁴ reported that normally pigmented black Africans responded differently to 4% homatropine plus 4% phenylephrine eye drops than did albino Africans. The latter responded in a manner similar to that of white subjects (i.e., onset of dilation and peak dilation occurred more rapidly).

Many rabbits, but not all, contain enzymes absent in humans that are capable of hydrolyzing and inactivating muscarinic antagonists. Atropinase (atropinesterase) is one of these and may protect rabbits from the toxic effects of belladonna ingested during leaf eating. Atropinase is also found in some strains of Pseudomonas.⁶⁶⁵ Its existence in mammalian species other than rabbits (e.g., rats and guinea pigs) is not well documented.⁶⁶⁶

Cauthen and colleagues⁶⁶⁷ found that rabbit atropinase hydrolyzed atropine and scopolamine but not cocaine. Although all three were esters and had similar structures, a different enzyme inactivated cocaine (Fig. 11). Cocainesterase did not hydrolyze atropine or scopolamine. The effects of both atropinase and pigment binding were studied by Salazar and co-workers.⁶⁶³ In rabbits with atropinase, the molecules bound to pigment were atropine and not its hydrolysis products, tropic acid and tropine. They found that the half-times for recovery of rabbits from atropine mydriasis were: albino rabbits with atropinase, 3.8 hours; albino rabbits without atropinase, 29.7 hours; pigmented rabbits with atropinase, 12.4 hours; and pigmented rabbits without atropinase, 96 hours or more.

Hemicholinium is a cholinergic blocking agent that exerts its effect at a site other than the receptor. It prevents uptake of choline by nerve endings. This results in decreased acetylcholine synthesis. It is highly toxic because it prevents acetylcholine synthesis by both nicotinic and muscarinic neurons. Its antinicotinic action results in paralysis of the respiratory muscles and death. Rabbits and cats can be killed from the systemic absorption of three drops of 1% hemicholinium.⁶⁶⁸ At concentrations below 0.5%, single drops have no effect. At higher concentrations, a single drop instilled unilaterally will produce pupillary dilation bilaterally and respiratory embarrassment within 40 minutes. If 0.001% to 0.0035% benzalkonium is added to 0.25% to 0.50% hemicholinium solutions, sufficient drug is absorbed through the cornea to produce dilation unilaterally without respiratory complications. The pupil does not respond to light or to stimulation of the ciliary nerve. These effects occur within 60 minutes and are usually gone within 6 hours. Hemicholinium is approximately 0.01 as potent a mydriatic agent as atropine (e.g., in mice, 0.1% hemicholinium gives as much dilation as 0.001% atropine sulfate). Topical 0.5% hemicholinium lowers the intraocular pressure in most, but not all, cats. There is an associated decrease in outflow facility. Therefore, the presumed mechanism of the hypotensive effect is a large decrease in aqueous humor production. Subconjunctival and intracameral injections of hemicholinium have produced corneal anesthesia in experimental animals.^669,670 This may indicate a role for the corneal epithelial cholinergic system in touch-pain sensation.

OCULAR PHARMACOLOGY. Changes in pupillary diameter are more easily monitored than are changes in lens accommodation. As a result, animal studies of muscarinic antagonists have concentrated on the former. Gordon and Ehrenberg⁴⁸⁶ demonstrated that dilation from muscarinic antagonists is due to a parasympatholytic effect. The eyes of anesthetized albino rabbits, dilated with cyclopentolate or atropine, no longer responded to third-nerve stimulation but continued to dilate further on stimulation of cervical sympathetic neurons. Lund Karlsen,¹⁹² using quinuclidinyl benzilate, which is a muscarinic antagonist with 10 times higher affinity for the receptor than atropine,^671,672 demonstrated that muscarinic receptors are found only in the sphincter region of the iris. Denervation did not increase their number. In one study, kittens and cats received daily topical atropine drops for 13 weeks.⁶⁷³ One year later there was a relative miosis and an eightfold increase in the number of muscarinic iris receptors in the kitten eyes, whereas adult cat eyes were unchanged from contralateral untreated eyes. Smith⁶⁵³ dilated the eyes of a volunteer with cyclopentolate before and after guanethidine drops. The pupil diameter after guanethidine paralysis of the dilator muscle was 7.05 mm, only slightly less than that before guanethidine (7.75 mm). This was consistent with the sphincter muscle playing the dominant role in determining the pupil size. Korczyn and Laor,⁶⁷⁴ after observing the pupillary responses of a patient with Waardenburg's syndrome given different pharmacologic agents (atropine, homatropine, guanethidine, and cocaine), suggested that atropine might have a partial direct α-agonistic action in addition to being antimuscarinic.

Both muscarinic agonists and antagonists, at concentrations that are submaximally effective, alter the pupillary light reflex in a similar manner: the speed and extent of the light response are diminished.³⁸³ This suggests that both types of drugs limit the number of muscarinic receptors on the sphincter muscle available for constriction to endogenously released acetylcholine. After a muscarinic antagonist is given, the pupils dilate with a speed proportional to the difference between the maximum pupil diameter and the actual pupil diameter.⁶⁷⁵

Accommodation has been considered a cause of myopia. Monkeys forced to do only near work develop myopia.⁶⁷⁶ Preventing accommodation with the use of atropine arrests the development of myopia in experimental animals.⁶⁷⁷ However, atropine may be working at other sites, either within the eye or as a result of systemic absorption. For example, pituitary secretion of growth hormone is inhibited by low blood levels of atropine. Subcutaneous atropine injections prevent chick eyes from developing myopia even though their ciliary muscles have nicotinic receptors and would not be expected to be affected by the drug.⁶⁷⁸ Furthermore, myopia can occur in animals whose ability to accommodate has been destroyed by lesions in the Edinger-Westphal nuclei⁶⁷⁹ and in animals who are normally unable to accommodate.⁶⁸⁰ Daily administration of the relatively selective M₁ antagonist pirenzepine prevents chick myopia,^681,682 and alternating daily between pirenzepine eye drops and pirenzepine subcutaneous injections prevents myopia in a mammal, the tree shrew.⁶⁸³ Because mammalian ciliary muscle contains primarily M₃ receptors, this effect of pirenzepine implies a site of action other than on the muscle producing accommodation. However, the relatively large doses of pirenzepine applied could result in a loss of M₁ receptor selectivity or in a toxic effect.

Monkeys treated unilaterally twice a day with 1% atropine eyedrops from age 10 days to 8 months developed an anisometropic amblyopia that remained present 4 months after discontinuation of the drug.⁶⁸⁴

The corneal epithelium contains acetylcholine. Its physiologic role is unknown. Umrath and Mussbichler⁶⁸⁵ and von Oer⁶⁸⁶ found that atropine drops caused a relative corneal anesthesia in rabbits. Atropine is structurally similar to the anesthetic cocaine. However, van Alphen,¹⁵⁵ studying both rabbits and humans, could not confirm that atropine produced anesthesia.

THERAPEUTICS. Mydriasis. Mydriasis aids the visualization of those intraocular structures behind the iris. The amount of mydriasis produced by a drug can be altered by the light reflex and by intraocular inflammation. α-Adrenergic agonists, such as phenylephrine, produce mydriasis by stimulating the iris dilator muscle. However, much of this dilation is lost when the retina is stimulated by a bright light (e.g., that of an ophthalmoscope or slit lamp) because of sphincter muscle constriction. When muscarinic antagonists produce mydriasis, light-induced relaxation of the dilator muscle does not result in a significant decrease in pupillary diameter. Most clinical studies of mydriatic agents do not measure pupil size in response to bright lighting. This is an especial failing when the mydriasis from an α-adrenergic agonist is being compared with that from a muscarinic antagonist.

Mydriasis also plays a major role in preventing posterior synechiae to the anterior lens capsule. The anterior lens surface is convex (Fig. 12). As mydriasis increases, the area of posterior iris surface in contact with the lens capsule decreases. Cycloplegia is of additional value because it reduces both the convexity and the thickness of the lens. If posterior synechiae do develop while the iris is dilated, there is less chance of iris bombé (as the pupil becomes more dilated, more synechiae must form if aqueous humor flow is to be blocked). This relationship is expressed by πd, where d is the diameter of the pupil. There is some controversy as to whether a mobile iris is or is not superior to a dilated fixed iris when intraocular inflammation is present.⁶⁸⁷ The rationale of those who believe mobility is superior is that the base of the dilated iris is thicker and may be in contact with the trabeculum. Prolonged mydriasis may, therefore, lead to anterior synechiae. A mobile iris would effectively prevent both anterior and posterior synechiae.

The results from investigations of mydriatic efficacy will be presented individually. Comparisons between studies are virtually impossible because different brands, concentrations, numbers of instillations, and drop intervals are used. In addition, patient populations are not uniform in age, race, and iris pigmentation. If one generalization does emerge, it is that all the commercially available muscarinic blocking agents produce adequate mydriasis if given either frequently enough or in high enough concentration. Eucatropine holds a unique position. Its muscarinic blocking activity is sufficient to produce mydriasis but too weak to produce significant cycloplegia. It would be an ideal agent when mydriasis alone is desired were it not for its prolonged latency, especially in darker irides. In these, it is not uncommon for maximum mydriasis to require several drops and well over 1 hour.

The efficacy of 1% atropine drops three times a day for 3 days was demonstrated by Marron⁶⁸⁸ in subjects 15 to 40 years of age. He noted that 5 minutes after the first drop there was a mean decrease in pupil diameter of 0.6 mm. By 40 minutes, maximum mydriasis had been achieved. In 16 of 107 subjects, maximum mydriasis was no longer present by the end of the second day or the beginning of the third day. However, compliance was not controlled, and these subjects may not have taken all their drops. In general, 4 to 6 days after the last drop, the pupils began to react to light, but 12 days were required for full recovery. In 21 subjects, with a mean age of 23 years, a single drop of 0.5% scopolamine also produced an initial miosis. By 20 minutes, maximum mydriasis had occurred. This lasted at least 90 minutes. Full recovery occurred by the eighth day. Early miosis also occurred when pupils were dilated with 5% homatropine, followed in 5 minutes by 1% hydroxyamphetamine, followed in 5 minutes by a second drop of homatropine. Maximum mydriasis occurred by 30 minutes and remained for 1 hour. Pupillary diameters were normal in 48 hours.

Wolf and Hodge⁶⁸⁹ compared single drops of 1% atropine, 1% methylatropine, and 1% homatropine in subjects 16 to 37 years of age. The respective times until maximum mydriasis were 40, 50, and 40 minutes. However, the amounts of maximum mydriasis differed, the respective ratios being 1.4:1.3:1. The onset of recovery for all three drugs was 6 hours.

Milder and Riffenburgh⁶⁹⁰ compared 0.5% cyclopentolate (two drops, each given 10 minutes apart to one eye) with 5% homatropine (two drops, each given 10 minutes apart to the other eye). The eye receiving homatropine also received a single drop of 1% hydroxyamphetamine. Cyclopentolate produced dilation sooner, but the mean maximum mydriasis achieved with the homatropine-hydroxyamphetamine combination was greater: 6.7 mm for the former versus 7.4 mm for the latter. Recovery also occurred sooner with cyclopentolate, often beginning by 3 hours and usually not complete until the next day. The mydriatic effect was less in black subjects in both magnitude and duration.

Gordon and Ehrenberg⁴⁸⁶ gave two drops of 0.5% cyclopentolate followed by a third drop 5 minutes later. Maximum mydriasis occurred at approximately 30 minutes, and the mean final pupillary diameter (7 mm) was greater than that achieved by 2% homatropine (6.1 mm) given in a similar manner. Recovery occurred within 20 hours of cyclopentolate administration but required up to 48 hours with homatropine administration.

Two drops of 1% cyclopentolate were administered to 48 children whose mean age was 7 years.⁶⁹¹ Instilling the drops one minute apart produced as much mydriasis as instilling them 5 minutes apart.

Five volunteers were given (at different times) one drop of each of four different muscarinic antagonists in pH 6.5 physiologic saline: 5% eucatropine, 0.5% tropicamide, 0.5% homatropine, and 0.5% cyclopentolate.⁶⁹² At 20 minutes postdrop, the mean mydriatic effects, compared with baseline, were: eucatropine (1.19 mm) < homatropine (1.99 mm) < tropicamide (3.72 mm) < cyclopentolate (3.96 mm). The maximum mydriasis was in the same order: 2.89 mm, 3.80 mm, 4.02 mm, and 4.42 mm. Percent recovery at 8 hours was: tropicamide (95%) > eucatropine (72%) > homatropine (30%) > cyclopentolate (12%). At 24 hours, recoveries from eucatropine and tropicamide were essentially complete, but not from homatropine (79%) or cyclopentolate (44%).

Merrill and colleagues⁶⁹³ found that 0.5% or 1% tropicamide almost always produced maximum mydriasis within 30 minutes, whereas 1% cyclopentolate, 5% homatropine, or 10% phenylephrine almost always required 60 to 90 minutes. The magnitude of dilation from tropicamide at 30 minutes was greater than that from the other drugs at 60 minutes. Tropicamide 1% was more effective than 0.5% tropicamide. Mydriasis disappeared 6 hours after 1% tropicamide in a group of 17 white subjects, aged 8 to 20 years; however, mydriasis from 1% cyclopentolate was still present in 82% of these subjects.

Gettes⁴⁸⁵ found single drops of 4% cocaine and 0.05% tropicamide about equal in mydriatic potency. So, too, were 1% hydroxyamphetamine and 0.1% tropicamide when compared 30 minutes postinstillation. Indirect ophthalmoscopy could be performed after a single drop of 0.5% tropicamide or after a combination of 0.2% tropicamide and 2% phenylephrine.

Gambill and associates⁶⁹⁴ compared the mydriatic effects of 0.5% tropicamide and 2% homatropine in 15 subjects using pupillography (Table 2). The maximum amount of mydriasis from tropicamide was approximately 5% greater than that from homatropine in eyes with light irides, and approximately 14% greater in eyes with dark irides.

TABLE 2. Comparisons of Mydriatic Effects of Tropicamide 0.5% and Homatropine 2%*

A single eyedrop containing tropicamide and phenylephrine in various combinations (i.e., 0.5% to 1% tropicamide and 2.5% to 10% phenylephrine) produces adequate but not maximum pupil dilation.^695–697

Carpel and Kalina⁶⁹⁸ dilated the eyes of 25 premature infants with birth weights of 800 to 2900 g. Three applications of 1% cyclopentolate, one drop in each eye every 5 minutes, were given, and the pupils were measured 30 minutes after the last drop. Then 10% phenylephrine was given to the right eye only and the pupils were remeasured 30 minutes after the last drop. The mean pupillary diameter after cyclopentolate alone was less than 70% of the corneal diameter at both readings. In the eyes receiving cyclopentolate and phenylephrine, the mean pupillary diameter increased to approximately 90% of corneal diameter.

Alzheimer's dementia is associated with degeneration of central nervous system cholinergic neurons. It is unknown if this is a primary or secondary finding. Because of this association, iris supersensitivity to tropicamide has been suggested as a diagnostic test. Pupil supersensitivity to a drop of 0.01% tropicamide was reported to differentiate non-Alzheimer patients from those who had the disease or were suspect.⁶⁹⁹ However, multiple studies have failed to confirm this finding.^700–702

Cycloplegia. Ciliary muscle paralysis prevents the eye from increasing the convex power of the lens. This occurs because the curvature of the intraocular lens cannot be altered. It is not that a change in the corneal curvature is prevented. This was shown by Daily and Coe,⁷⁰³ who performed keratometry readings before and after cycloplegia. The two were identical. Poinoosawmy and co-workers⁵¹⁸ reported that stimulation of accommodation with pilocarpine did alter corneal curvature.

With the use of ultrasound, ciliary body thickness was found to decrease by 0.06 mm 2 hours after volunteers received a drop of 1% cyclopentolate.³⁹³ This was believed to be due to relaxation of the ciliary muscle.

Cycloplegia is of clinical importance because it allows a baseline evaluation of the refractive status of the eye. In subjects capable of accommodation, a dynamic focusing system is present. As the young child looks from physician, to retinoscope light, to mother, to distant fixation target, the diopteric power of the intraocular lens changes. Accurate refraction becomes difficult or impossible. This is not acceptable in children with accommodative esotropia or anisometropic amblyopia. In orthotropic children with normally developing vision, it might be argued that cycloplegia is unnecessary because small errors in the measurements are unimportant. However, this defense assumes that the refractionist knows his or her errors are insignificant without having measured them. On the other hand, in younger subjects, cycloplegia will occasionally induce errors in the amounts and axes of astigmatism. These may result from the taut zonules tilting the lens in a nonphysiologic manner. The art of refraction lies in blending both cycloplegic and noncycloplegic findings.

The assessment of the relative cycloplegic activity of different drugs suffers from the same limitations mentioned for mydriatic activity. Different concentrations and commercial preparations are used in populations that differ in race, age, and iris pigmentation. Furthermore, the techniques for determining residual accommodation differ from paper to paper. Some measure the near point of accommodation with print or a Prince rule; others use lens techniques that incorporate near-point determinations; and others use retinoscopic findings. If blur-point techniques are used to measure residual accommodation, then depth of focus is being evaluated as well.

Atropine (1%) drops, three times a day for 3 days, in 107 subjects ranging in age from 15 to 40 years produced maximum cycloplegia 23 to 48 hours after the first drop. Accommodation returned rapidly enough that most subjects could read newsprint 3 days after the last drop. Giving drops five times a day did not increase cycloplegia.⁶⁸⁸ A single drop of 0.5% scopolamine in 21 subjects with a mean age of 23 years produced maximum cycloplegia in 40 minutes; this persisted for at least 90 minutes. By the third day, most could read. Normal accommodation returned within 10 days. Homatropine (5%), two applications given 5 minutes apart to 25 subjects with a mean age of 28 years, resulted in maximum cycloplegia 50 minutes after the first drop. This was maintained for 30 minutes. By 6 hours, subjects could read, and full return of accommodation occurred within 48 hours.

Atropine (1%), as an ointment, was given twice a day for 4 days to ⁶⁴⁸ 1-year-olds.⁴⁸⁹ The cycloplegia induced was compared with that from a single drop of 1% cyclopentolate given to each of 355 1-year-olds 30 minutes before retinoscopy. A third group of 415 1-year-olds received two instillations of 1% cyclopentolate, 10 minutes apart, with retinoscopy performed 30 minutes after the last drop. There were no significant differences in the results obtained from one or two drops of cyclopentolate. However, there was significantly more (p < 0.01) cycloplegia, averaging approximately 0.4 D per subject, in the group receiving atropine. Of esotropic children under age 5.5 years, 22% had 1 D or more of hyperopia uncovered by atropine rather than cyclopentolate.⁷⁰⁴

Single drops of 1% atropine, methylatropine, or homatropine were given to subjects 16 to 37 years of age. The times until maximum cycloplegia were 5 hours, 5 hours, and 25 minutes, respectively.⁶⁸⁹ However, the amounts of maximum cycloplegia were not the same for all three drugs. Residual accommodation was least after atropine and most after homatropine. The duration of maximum cycloplegia was 1 day for atropine, 6 hours for methylatropine, and 1 hour for homatropine. The times until complete cycloplegia recovery from atropine (9.2 ± 3.6 days) and from methylatropine (6.8 ± 3.8 days) were not significantly different.

Twelve black and 19 white subjects, whose ages ranged from 8 to 48 years, were given 0.5% cyclopentolate to one eye and 5% homatropine to the other. Two instillations of each drug, 10 minutes apart, produced no significant differences in residual accommodation. Cyclopentolate had a more rapid onset but shorter duration: 40% of cyclopentolate-treated eyes achieved maximum cycloplegia in less than 15 minutes; 22% of cyclopentolate-treated eyes required 45 minutes or more. By 24 hours, the cyclopentolate effect, but not that of homatropine, was gone. In 30% of eyes receiving cyclopentolate, maximum cycloplegia lasted less than or equal to 30 minutes. It was concluded that the optimum time for refraction was 45 minutes or less after 0.5% cyclopentolate was given in this manner.⁶⁹⁰ Blacks tended to have less cycloplegia from both drugs. In subjects initially given two drops of 0.5% cyclopentolate to the eye, followed by a third drop 5 minutes later, similar results were obtained.⁴⁸⁶ Maximum cycloplegia occurred 30 to 60 minutes after the drops. The residual accommodation 60 minutes after the cyclopentolate drops was less than that from 4% homatropine, 1.1 D versus 2.0 D. In both studies, cyclopentolate recovery occurred within 24 hours, whereas that from homatropine lagged.

Single drops of 1% cyclopentolate, 1% tropicamide, and 0.5% tropicamide, were compared with use of a Prince rule.⁶⁹³ Tropicamide (0.5%) produced much less cycloplegia. In subjects under 10 years of age, the mean residual accommodation 30 minutes after a 0.5%, or 1% tropicamide drop was, respectively, 4.22 or 0.35 D. The maximum cycloplegic effect from 1% tropicamide was greater than that from 1% cyclopentolate or 5% homatropine but of much shorter duration (Table 3). Six hours after instillation of 1% tropicamide, 90% of accommodation had returned, whereas 6 hours after instillation of cyclopentolate, 42% of accommodation had returned.

TABLE 3. Residual Accommodation (Prince Rule) in Subjects 4 to 9 Years of Age After a Single Drop

However, a study by Gettes⁴⁹⁰ found that the cycloplegia produced by a single instillation of 1% tropicamide was inadequate. In 53% of the eyes of subjects whose ages ranged from 14 to 35 years, more than 2.5 D of residual accommodation remained 20 to 25 minutes after drug application. In the 20- to 35-minute period postinstillation, 38% of eyes had more than 2.5 D residual accommodation. If a second drop of 1% tropicamide was given 5 to 25 minutes after the first, 100% of these subjects had less than 2.5 D of residual accommodation 20 to 25 minutes after the second drop. If refractions were performed up to 35 minutes after the second drop, 10% of eyes had more than 2.5 D of residual accommodation. The effect of 1% tropicamide, 1% cyclopentolate, or 4% homatropine-1% hydroxyamphetamine given to one eye was compared with the cycloplegic effect of one of the other two solutions placed in the contralateral eye. Two instillations of each medication were given, 5 minutes apart.⁷⁰⁵ Refractions were performed 20 to 40 minutes after the second drop. Residual accommodation was less than 2.5 D in 79% of the eyes of white subjects and 69% of the eyes of black subjects given tropicamide, 83% of the eyes of white subjects given cyclopentolate, and 51% of the eyes of white subjects and 36% of the eyes of black subjects given homatropine. By 40 to 60 minutes after the second drop, residual accommodation was more than 2.5 D in over half of the eyes receiving tropicamide. However, 91% of eyes receiving cyclopentolate and 59% of eyes receiving homatropine still had less than 2.5 D of residual accommodation.

Milder⁷⁰⁶ performed similar studies in a masked manner. Two instillations of one drop of 1% tropicamide, 1% cyclopentolate, or 5% homatropine were given to each eye of the subjects. Tropicamide drops were given 5 minutes apart and the others were given 10 minutes apart. Residual accommodations were measured at 30 and 60 minutes. The values 30 minutes after the tropicamide were compared with those 60 minutes after cyclopentolate or homatropine. In 80% of subjects, homatropine was superior to tropicamide, and in 92% of subjects, cyclopentolate was superior to tropicamide. The magnitude of residual accommodation was inversely related to age, and it was concluded that tropicamide should not be used for cycloplegia in patients under 40 years of age.

Miranda⁷⁰⁷ preceded 1% cyclopentolate drops to each eye with bilateral proparacaine anesthetic drops. In one eye, a second drop, 1% tropicamide, was given 25 seconds after the cyclopentolate. Refractions were performed 30 to 40 minutes later. Residual accommodation was measured by holding a reading card at 16 inches and gradually overminusing a + 2.50 lens. In a test group of 185 subjects ranging in age from 7 to 36 years, the cyclopentolate-tropicamide combination was superior in heavily pigmented eyes. In blue, green, light brown, and medium brown irides, the residual accommodation of the two eyes ranged from 0.25 to 1.00 D. However, the residual accommodation in eyes with dark brown irides receiving cyclopentolate alone was 1.25 to 5.50 D, whereas that in eyes receiving cyclopentolate-tropicamide was 0.50 to 1.25 D. Age was not as important a variable as was iris pigmentation. Equivalent cycloplegia was achieved in a group of children with a mean age of 7 years by instilling two drops of cyclopentolate 1 minute apart or 5 minutes apart.⁶⁹¹

Five volunteers were given, at different times, one drop of the following made up in pH 6.5 physiologic saline: 5% eucatropine, 0.5% homatropine, 0.5% tropicamide, and 0.5% cyclopentolate.⁶⁹² The mean maximum reduction in accommodation was eucatropine (2.85 D) < homatropine (6.17 D) < tropicamide (7.14 D) < cyclopentolate (8.64 D). The percent recoveries at 8 and 24 hours, respectively, were: eucatropine, 89% and 100%; tropicamide, 99% and 98%; homatropine, 39% and 89%; and cyclopentolate, 46% and 66%.

Analgesia. The source of pain during intraocular inflammation is unknown. The cornea is innervated by sensory fibers of the trigeminal nerve that pass near or through the uvea. In primates, trigeminal innervation is also present in the anterior stroma of the ciliary body and, to a lesser degree, in the iris and trabecular meshwork.^708,709 Cycloplegic agents relieve the discomfort associated with intraocular inflammation. The degree of relief is variable but often dramatic.

Malignant Glaucoma. Slack zonules may be a cause of malignant glaucoma. The lens comes forward and flattens the anterior chamber. This mechanism is resistant to peripheral iridectomy and muscarinic agonists. The latter may initiate or aggravate the condition. Chandler and Grant⁵³⁴ reported seven patients in whom malignant glaucoma developed after a peripheral iridectomy for narrow-angle glaucoma. Muscarinic antagonists, such as atropine, homatropine, and scopolamine, were of value in breaking these attacks. Presumably they acted by paralyzing the ciliary muscle, pulling the zonules taut, and drawing the lens posteriorly. Frezzotti and Gentili⁷¹⁰ found that long-term muscarinic antagonists also prevented recurrences of malignant glaucoma. However, Chandler and colleagues⁷¹¹ reported a more complex situation. Not all patients with malignant glaucoma responded to muscarinic antagonists. Those who did were not always controlled during long-term therapy. Although malignant glaucoma usually returned when muscarinic blocking agents were discontinued, one subject could be maintained on acetazolamide alone. Overall, their short-term success rate in 17 subjects was 60% and their long-term success rate was just under 50%.

Provocative Testing. Muscarinic antagonists can elevate intraocular pressure. Sometimes this is an undesired side effect. At other times, it is of diagnostic value.

CLOSED-ANGLE GLAUCOMA. Muscarinic antagonists have been used to predict which eyes with narrow angles are likely to develop acute closed-angle glaucoma. The rationale for this provocative action is that mydriasis thickens the iris base and occludes the angle. However, the results of testing are erratic. For example, Sugar⁷¹² instilled 4% homatropine into the eyes of four subjects who had had prior attacks of closed-angle glaucoma but who had not received iridectomies. Two did not have an increase in ocular pressure. The other two had marked elevations of more than 15 mmHg. In four narrow-angle glaucoma patients whose eyes were dilated with 10% phenylephrine, an adrenergic agonist, two had no increases in ocular pressure, one had an increase of 32 mmHg 30 minutes after instillation, and one had a latency of more than 5 hours before the pressure rose by 29 mmHg.

There appears to be little relation between the intrinsic muscarinic blocking potency of a drug and its ability to elicit a positive provocative test. Twenty subjects with prior histories of closed-angle glaucoma were tested. In 13 subjects, the drops were placed in the contralateral eyes (i.e., the eyes that had no histories of angle-closure attacks). In 7 subjects, the drops were placed in the diseased eyes.⁷¹³ Eucatropine (2%) did not elevate the ocular pressure of five eyes but did elevate the ocular pressure of five other eyes by a mean of 21 mmHg. Three of the five unresponsive eyes remained normotensive after 2% homatropine. The two eyes that did respond had ocular pressure increases of 36 and 37 mmHg. Eucatropine elevated the intraocular pressure in one eye that did not respond to homatropine. One of the five eyes unresponsive to eucatropine developed a marked elevation, of 42 mmHg, after 1% hydroxyamphetamine, an adrenergic agonist.

A decrease in tonographic outflow facility may occur more often than an increase in intraocular pressure.⁷¹⁴ Tonography was performed before and 1 hour after 5% eucatropine eyedrops were placed in 58 normotensive eyes with narrow angles. Twenty-eight percent had ocular pressure elevations of at least 8 mmHg, and 67% had outflow facility decreases of at least 25%. Of these 58 subjects, 55% eventually had a closed-angle attack. In retrospect, 44% had had a positive pressure provocative test and 88% had had a positive tonography provocative test.

CILIARY BLOCK GLAUCOMA. Ciliary block glaucoma occurs when the ciliary processes or ciliary muscle adhere to the back of the iris. This can occur spontaneously or after intraocular surgery (especially glaucoma filtering procedures). Muscarinic antagonists, such as atropine, can be of therapeutic value by paralyzing and flattening the ciliary muscle, thereby pulling away the adhering tissues.⁷¹⁵ The effectiveness of muscarinic antagonists is variable; chronic use may be necessary.

OPEN-ANGLE GLAUCOMA. Harris⁷¹⁶ observed that 2% of the “normal” open-angle population had a pressure elevation of 6 mmHg or more after topical applications of 1% cyclopentolate. Homatropine (5%) and 1% atropine were as effective as 1% cyclopentolate in provoking this response, but 0.5% tropicamide, 0.2% cyclopentolate, 5% eucatropine, and 10% phenylephrine were not. In approximately 70% of subjects responding to 1% cyclopentolate, 0.25% scopolamine was equally effective. Harris raised the question of whether this 2% of the population was or was not at risk of developing open-angle glaucoma. Subsequently, 58 subjects with a family history of open-angle glaucoma were studied.⁷¹⁷ In none did 1% cyclopentolate elevate the intraocular pressure more than 5 mmHg. This seemed to indicate that muscarinic antagonists were of little predictive value. A different approach was then investigated. Dexamethasone (0.1%) was instilled three times a day for 3 weeks by these 58 subjects. In those with a steroid-induced pressure elevation of at least 6 mmHg, 41% had an additional elevation of at least 6 mmHg after topical application of 1% cyclopentolate. In those without a steroid-induced pressure elevation, only 6% had an elevation of this magnitude after cyclopentolate. A positive cyclopentolate test correlated significantly with a steroid-induced reduction in tonographic outflow facility but not with a steroid-induced elevation in intraocular pressure. The importance of these observations with respect to predicting future cases of open-angle glaucoma remains unclear.

A second use of provocative testing has been to anticipate which open-angle glaucoma patients given systemic muscarinic antagonists will experience increases in their intraocular pressures. Medications with muscarinic blocking activity are given for ulcer therapy and psychiatric conditions, for example. Lazenby and associates^718,719 studied 24 subjects who had been off glaucoma therapy 3 or more days and 4 subjects who had been continued on medication. They found that topical 1% cyclopentolate caused a rise of 5 mmHg within 2 hours in half of these patients. Neither the cyclopentolate-responding group nor the cyclopentolate-nonresponding group had ocular pressure elevations during short-term oral atropine sulfate therapy. The atropine sulfate was given in two doses of 0.6 mg, 4 hours apart. A second group of 21 patients with open angles and elevated pressures was given oral atropine sulfate, 0.6 mg, three times a day for 7 days. Nine had responded to prior cyclopentolate eyedrops with an ocular pressure increase of 5 mmHg or more. In 4 of these 9, oral atropine was associated with an elevation in ocular pressure of 6 to 14 mmHg. In 1 of the 4, the pressure in one eye was elevated 9 mmHg and the pressure in the other eye was lowered 2 mmHg. In the remaining 5 cyclopentolate responders, the change in ocular pressure ranged from -2 to + 3 mmHg. A consistent relationship between the ocular pressure response and the tonographic outflow facility response was not observed (e.g., in 1 subject whose ocular pressures increased by 14 and 6 mmHg, the outflow facility values decreased from 0.08 μL/min/mmHg to 0.06 μL/min/mmHg in the former and increased from 0.08 μL/min/mmHg to 0.13 μL/min/mmHg in the latter). The 12 subjects who did not respond to cyclopentolate had little response to oral atropine. Their mean ocular pressures and outflow facilities before and after atropine were, respectively, 22.6 and 22.5 mmHg and 0.18 μL/min/mmHg and 0.19 μL/min/mmHg. These authors concluded that pretesting open-angle glaucoma patients with 1% cyclopentolate eyedrops often identified those at risk of developing further elevations in their ocular pressures from oral anticholinergic medications.

Myopia. It has been suggested that myopia results from accommodation (i.e., ciliary muscle contraction produces elongation of the eye). If so, then muscarinic antagonists, such as atropine, would prevent myopia by putting the ciliary muscle to rest. The evidence is, at best, suggestive. Dyer⁷²⁰ instilled 1% atropine once each day bilaterally in 86 children for 2 to 8 years. Nineteen percent had an increase in myopia of 1 D or more. In another group of 86 children given their full corrections, 84% had an increase of 1 D or more. Bedrossian⁷²¹ reported that children using 1% atropine for 1 year in one eye had a mean decrease in myopia of 0.21 D in that eye compared with an increase of 0.82 D in the control eye. However, the comparison was made by refracting the control eye with tropicamide. The data may only represent a difference in the cycloplegic potency of atropine versus tropicamide. Sampson⁷²² found that atropine drops seemed to prevent the progression of myopia, but on discontinuing the drops only 12% of subjects maintained their improvement for more than 6 months.

If muscarinic antagonists prevent myopia, then muscarinic agonists, by facilitating accommodation, might induce myopia. However, there are no reports of rapidly developing myopia in children with accommodative esotropia treated with DFP or echothiophate.

Perhaps atropine prevents myopia in a manner that is unrelated to the drug's effect on accommodation. Oral doses of 0.6 mg atropine abolish sleep-associated growth hormone secretion.⁷²³ Two 50-μL eyedrops of 1% atropine contain 1 mg atropine, an amount sufficient to have systemic effects.

Amblyopia. As an alternative to occlusive patching of the normal eye, disuse amblyopia can be treated with muscarinic antagonists. The blurring produced by cycloplegia penalizes the eye with normal acuity and encourages use of the amblyopic eye. This form of therapy will succeed only if the reduction in acuity of the normal eye exceeds that of the amblyopic eye. In cases of latent nystagmus, pharmacologic penalization may be the preferred treatment. In patients with eccentric fixation, penalization has been said to be contraindicated.⁷²⁴ However, others have reported dramatic success with this condition.⁷²⁵ Johnson and Antuna⁷²⁶ preferred using atropine in the ointment form because they believed any additional blurring from this viscous material was beneficial.

Single daily instillations of 1% atropine may be inadequate, and multiple drops may have to be given.⁷²⁷ Von Noorden and Milam⁷²⁵ used only one drop, given each morning, and reported good results. Successful therapy, defined as an improvement of two lines or more in visual acuity, occurred in 10 of 17 amblyopic eyes. Pharmacologic penalization not only improved vision but was also used to maintain vision. In 10 of 13 patients whose vision deteriorated when patching was discontinued, vision was maintained within one line of the best acuity by the use of atropine.

Penalization during that period of visual development can result in the initially normal seeing eye developing amblyopia.⁷²⁸ This would occur after the initially amblyopic eye had improved to the point that its acuity surpassed that of the contralateral atropine-blurred eye. Ikeda and Tremain⁷²⁹ studied this phenomenon in experimental animals. The eyes of kittens were chronically blurred with atropine drops, unilaterally or bilaterally. When these cats reached maturity, single-cell recordings were made from their lateral geniculate bodies and visual cortices. Lateral geniculate body recordings were abnormal in unilaterally and bilaterally treated cats. Visual cortex recordings were abnormal only in unilaterally treated cats.

OCULAR HYPERTENSION FROM TOPICAL THERAPY. Muscarinic antagonists given topically or systemically can elevate intraocular pressure. This occurs in normotensive subjects and glaucoma patients whether the angles are open or narrow. The mechanisms involved are varied and not completely understood.

Pigment Release. Mobility of the iris is associated with release of uveal pigment and a transient block of the trabecular meshwork. Mitsui and Takagi⁷³⁰ removed aqueous humor from subjects diagnosed clinically as releasing pigment; the material was identified as being compatible with uveal melanin. They challenged subjects with 5% phenylephrine, 1% atropine, 1% homatropine, 0.5% physostigmine, and 1% pilocarpine. Phenylephrine was by far the most effective in releasing pigment. The other drugs were about equal in effectiveness, with the exception of pilocarpine, which elicited pigment release in only 1 of 16 subjects. Calhoun⁷³¹ dilated the eyes of subjects having Krukenberg spindles with 2% homatropine plus 10% phenylephrine. The presence or absence of an increase in intraocular pressure correlated with the amount of pigment released into the aqueous humor. Valle⁷³² studied 21 eyes that had or were suspected of having untreated open-angle glaucoma and that responded to a drop of 1% cyclopentolate with intraocular pressure elevations of 8 to 20 mmHg. These pressure responses were maximum 1 to 4 hours after instillation of cyclopentolate. By 6 hours, they approached normal values. In one third of the eyes, pigment was liberated into the anterior chamber. The degree of pressure elevation correlated with the amount of pigment released.

Pseudoexfoliation. A 1% cyclopentolate provocative test was performed on Scandinavian patients suspected of having open-angle glaucoma.⁷³³ An increase of 8 mmHg in intraocular pressure was considered positive. Of the 8% of patients having a positive test, half had pseudoexfoliation of the lens capsule.

Open-Angle Glaucoma. The response of eyes with open-angle glaucoma to muscarinic antagonists is not entirely predictable. Some eyes develop elevations of intraocular pressure and some do not. Of 15 eyes with open-angle glaucoma that were dilated with two drops of 5% homatropine given 10 minutes apart, 8 had intraocular pressure elevations of 6 to 13 mmHg, 4 had elevations of less than 6 mmHg, 2 did not change, and 1 had a decrease in ocular pressure of 3 mmHg.⁷³⁴ In a comparison of 5% homatropine, 2% hydroxyamphetamine, and a solution containing both, the mean ocular pressure rise from the combination was the largest. It occurred 3 hours after instillation and averaged 5.6 mmHg. The maximum homatropine and hydroxyamphetamine responses were at 2 hours and averaged 4.5 and 3 mmHg, respectively. Sugar⁷¹² instilled 4% homatropine in one eye and 10% phenylephrine in the other eye of 51 open-angle glaucoma subjects who had not taken their medications that day. The changes in intraocular pressures were determined 30 and 60 minutes later. The range of responses to homatropine was -4 to + 8 mmHg and the range of responses to phenylephrine was -8 to + 8 mmHg. Galin⁷³⁵ dilated bilaterally the eyes of three subjects with open-angle glaucoma off medication and six normotensive subjects with open angles; he used 1% cyclopentolate, every 15 minutes for four doses. In seven of the subjects, an increase in intraocular pressure of 8 mmHg or more occurred, but only unilaterally.

Harris and Galin⁷³⁶ studied 69 open-angle glaucoma patients receiving either bilateral pilocarpine or bilateral echothiophate therapy. Each subject was challenged with three drops of 1% cyclopentolate unilaterally. The drops were given 15 minutes apart. The other eye served as control. Forty-one percent of 53 pilocarpine-treated eyes and 50% of 16 echothiophate-treated eyes responded to cyclopentolate with pressure elevations of 6 mmHg or more. It was not unexpected that muscarinic antagonists would reverse the hypotensive effects of muscarinic agonists, but it was surprising that the reversal did not occur in all eyes. Thirty-eight of these patients were then taken off muscarinic therapy for 2 weeks and rechallenged. Twenty-four percent had an increase in intraocular pressure of 6 mmHg or more. This incidence was lower than that while on therapy, probably because the pressures were already near their peak before they were challenged. Combining these results with those of prior studies, the authors stated that the overall incidence of a 6-mmHg or greater ocular pressure elevation after challenge by cyclopentolate drops was 33% for open-angle glaucoma subjects on muscarinic agonist therapy.

Valle⁷³⁷ compared the cyclopentolate response of glaucoma suspects with intraocular pressures of 22 to 24 mmHg with that of untreated open-angle glaucoma patients. Two drops of cyclopentolate were given, each 15 minutes apart, and the ocular pressures were followed for a minimum of 3 hours. In the suspect group, the mean increase in intraocular pressure was 0.4 ± 2.5 mmHg. This was significantly less than that of the glaucoma group (2.5 ± 3.1 mmHg). Only 2% of the suspect group and 9% of the glaucoma group had elevations in pressure of 8 mmHg or more. There was no correlation between the initial ocular pressure and the magnitude of the pressure response.

The eyes of patients with primary open-angle glaucoma were dilated with 1% tropicamide plus phenylephrine.⁷³⁸ The mean ± SD intraocular pressure before dilation was 22.9 ± 6.1 mmHg. One hour after the drops, the mean intraocular pressure was 25.8 ± 7.7 mmHg. Individual changes in ocular pressure ranged from -6 mmHg to + 22 mmHg. An intraocular pressure increase occurred in 60% of subjects and a decrease in 30%. Increases of 10 mmHg or more occurred in 12% of subjects, and increases of 20 mmHg or more occurred in 2%. The only significant risk factor for an elevation was the therapeutic use of muscarinic agonists: 41% of these subjects had an elevation of 5 mmHg or more and 19% had an elevation of 10 mmHg or more. There was no significant correlation between the intraocular pressure rise after dilation and the intraocular pressure rise after laser trabeculoplasty.

The tonographic outflow facility responses to muscarinic antagonists may be more consistent than the ocular pressure responses. Galin⁷³⁵ gave multiple drops of cyclopentolate to 18 eyes. Nine developed elevations in intraocular pressure of 8 mmHg or more and 11 developed decreases in outflow facility of 25% or more. Schimek and Lieberman⁷³⁹ found that all 17 eyes of open-angle glaucoma subjects receiving cyclopentolate had decreases in outflow facility, whereas only 15 eyes had increases in intraocular pressure. Christensen and Pearce⁷⁴⁰ gave two drops of 5% homatropine unilaterally to 35 chronic simple glaucoma patients. The mean increase in ocular pressure was 3 mmHg, with a range of -2 to + 19 mmHg. The mean decrease in tonographic outflow facility was 28%.

Normotensive Subjects With Open Angles. One eye of each of 70 normotensive subjects, ranging in age from 22 to 80 years, was dilated with 5% homatropine. The ocular pressure change 45 minutes later was similar to that of the control eye, ranging from -6 mmHg to + 3.5 mmHg.⁷⁴⁰ Tonographic outflow facilities were reduced in the challenged eyes 9% to 10% more than in the control eyes. However, Harris⁷¹⁶ found a 2% incidence of a 6-mmHg or greater pressure elevation in 550 normotensive open-angle subjects after 1% cyclopentolate, one drop every 15 minutes for three doses. Homatropine (5%) and 1% atropine were as effective as cyclopentolate in elevating the ocular pressure.

Chronic Narrow-Angle Glaucoma. Lowe⁷⁴¹ found that many subjects with chronic narrow-angle glaucoma and open peripheral iridectomies developed angle closure, increased intraocular pressure, and decreased outflow facility after their eyes were dilated with homatropine. Phenylephrine (10%) dilatation did not produce these changes. The explanation offered was that the pull of the sphincter muscle against the phenylephrine-stimulated dilator muscle produced a vector of force pulling the iris base away from the angle. This, presumably, did not occur after homatropine. Harris and Galin⁷⁴² found similar results. Dilating 26 eyes successfully treated with peripheral iridectomies (i.e., none had residual chronic closed-angle glaucoma) did not produce elevations in ocular pressures. However, in seven of eight eyes with chronic narrow-angle glaucoma, 1% cyclopentolate, but not 10% phenylephrine, produced pressure elevations of 6 mmHg or more. They did not confirm Lowe's⁷⁴¹ explanation because the filtration angles, as shown by gonioscopy, remained open in the seven eyes developing elevated intraocular pressures after homatropine.

Acute Closed-Angle Glaucoma. Only 1 of 250 primary care physicians routinely dilates pupils, even when examining diabetics.⁷⁴³ The stated excuse is the fear of provoking acute narrow-angle glaucoma.⁷⁴⁴ However, if patients with a history of glaucoma or shallow anterior chambers on penlight examination are excluded, there is less than a 0.3% chance of inadvertently dilating the eyes of a subject with potentially occludable filtration angles, as determined by gonioscopy.⁷⁴⁵

The mechanisms by which muscarinic antagonists produce acute closed-angle glaucoma were stated earlier. However, the incidence of pharmacologically induced angle closure in eyes without previous attacks is unknown. Therefore, a controversy exists. Some ophthalmologists will not perform a dilated intraocular examination on subjects with narrow angles for fear of provoking an acute attack of glaucoma. Other ophthalmologists routinely instill muscarinic antagonists because they believe that the incidence of acute angle closure is lower than the incidences of other types of ocular pathology (e.g., retinal tears, retinal detachments, and melanomas) that would otherwise escape detection. It can also be argued that pharmacologically provoking an attack of angle closure is in the patient's best interest. The ophthalmologist can make the correct diagnosis and provide adequate treatment. Spontaneous attacks that occur without medical supervision are more likely to lead to severe visual loss and intractable glaucoma.

OCULAR HYPERTENSION FROM SYSTEMIC THERAPY. Lund Karlsen¹⁹² gave guinea pigs intraperitoneal injections of atropine at a concentration that was lethal in humans (i.e., 0.225 mg/kg). This produced iris concentrations that were only 3% of those achieved by eyedrops containing 0.01 mg atropine. This demonstrates why systemic muscarinic antagonists do not commonly affect intraocular structures (i.e., relatively small amounts enter the eye). The ensuing discussion deals with the ocular effects of systemically administered muscarinic antagonists. Normotensive, open-angle glaucoma, and narrow-angle glaucoma subjects are considered separately. The responses are variable within all three groups. Most subjects demonstrate little or no effect. However, a minority do respond significantly. As a result, the ophthalmologist cannot dismiss a priori systemic anticholinergic medications as being of little consequence.

Normotensive, open-angle subjects were given intramuscular injections of 0.6 to 0.8 mg atropine or 0.4 to 0.6 mg scopolamine. Three of the eight receiving atropine developed 0.5 to 1.5 mm mydriasis, and seven of the eight receiving scopolamine developed 0.5 to 2 mm mydriasis.⁵⁹⁰ There was a mean increase of 8 mm in the near point of accommodation after atropine and a mean increase of 27 mm after scopolamine. When 100 normotensive subjects ranging in age from 16 to 40 years were given atropine 0.016 mg/kg, 13 developed pupillary dilations of 2 mm or less and 9 developed ocular pressure elevations of 3 mmHg or less.⁷⁴⁶ Similar results were obtained after intramuscular injection of scopolamine 0.01 mg/kg.⁷⁴⁷ Cozanitis and co-workers⁷⁴⁸ reported that atropine 0.01 mg/kg did not affect the pupillary diameter.

A single intramuscular injection of atropine sulfate, 2 mg, in 13 male normotensive subjects produced a mean maximum pupil dilation of 1.25 mm at 2 hours postinjection.⁷⁴⁹ By 5 hours postinjection this effect was gone. Bye and colleagues⁷⁵⁰ used pupillography to study the effects of oral atropine sulfate, 0.02 mg/kg. During the 3 hours after ingestion, the mean pupillary diameter increased significantly, going from 7.26 mm to 7.49 mm. The amplitude of the light reflex was significantly diminished. Other anticholinergic drugs that have been studied include oral propantheline⁷⁵¹ and intramuscular glycopyrrolate.⁷⁴⁸ Propantheline, 15 mg every 6 hours, was given for 24 hours. There was a 16% incidence of an ocular pressure elevation of at least 4 mmHg and a 12% incidence of an ocular pressure depression of at least 4 mmHg. The incidences of pupillary dilation or constriction of at least 1 mm were similar. Glycopyrrolate (glycopyrronium), 0.04 mg/kg, did not affect either the pupillary diameters or the ocular pressures of the 10 subjects who were tested.

Eight open-angle glaucoma patients off therapy for 48 hours were given either 0.5 to 0.6 mg atropine or 0.4 to 1 mg scopolamine intramuscularly. Usually the intraocular pressures fell or were unchanged. In one eye there was a pressure increase of 3 mmHg after atropine, and in two eyes there were pressure increases of less than 2 mmHg after scopolamine.⁷⁵² Thirty-four eyes with open-angle glaucoma were maintained on therapy, and atropine 0.01 mg/kg was injected intramuscularly. Pupillary diameters, ocular pressures, and accommodation were followed for 2 hours.⁷⁵³ The intraocular pressures of 22 eyes were unchanged or were reduced. In the remaining 12 eyes, the ocular pressures increased by 2 to 5 mmHg. Overall, neither the ocular pressure changes nor the pupil diameter changes were significant. Ten glaucoma subjects were given scopolamine 0.01 mg/kg. There were no significant changes in ocular pressure or pupil size.⁷⁴⁷ The ocular pressures of 94 eyes with untreated open-angle glaucoma were monitored while propantheline, 15 mg every 6 hours, was administered for 24 hours. In 21% of these eyes there was an increase in ocular pressure of 4 mmHg or more, and in 10% there was a decrease of 4 mmHg or more. In one eye, the ocular pressure elevation was 16 mmHg. Pupillary dilations were always less than 1 mm.⁷⁵¹

Narrow-angle glaucoma patients were studied by Schwartz and associates.⁷⁵² Six subjects, normotensive at the time of injection, were given 0.5 to 0.6 mg atropine or 0.4 to 1 mg scopolamine intramuscularly. Five of the six had unilateral iridectomies. The mean elevation in intraocular pressure was less than 0.5 mmHg. Six subjects with previous histories of closed-angle attacks received intramuscular injections of atropine 0.01 mg/kg.⁷⁵³ The ocular pressures remained unchanged or decreased. When seven narrow-angle glaucoma patients were given propantheline, 15 mg every 6 hours for 24 hours, 21% of the 14 eyes had increases in ocular pressure of 4 mmHg or more. In two eyes, the pressure elevations were 16 and 17 mmHg. In none of the 14 eyes did the pupils dilate more than 1 mm.⁷⁵¹

SYSTEMIC TOXICITY FROM TOPICAL THERAPY. Toxicities have been reported after topical muscarinic antagonist therapy and from the ingestion of eyedrop solutions.⁷⁵⁴ All the commonly used blocking agents have been implicated.

Eyedrops produce measurable drug levels in the blood. A single 40-μL drop of 1% atropine given unilaterally to recumbent patients produced a mean ± SD peak plasma concentration of 860 ± 402 pg/mL within 8 minutes.⁷⁵⁵ A single 35-μL drop of cyclopentolate given unilaterally to supine children whose mean age was 9 ± 3 years produced a maximum plasma level of 2.2 ± 1.6 ng/mL at 5 minutes.⁷⁵⁶ Two 30-μL drops of 1% cyclopentolate produced a mean peak plasma concentration of approximately 3 ng/mL within 30 minutes; in some subjects a second peak occurred at 2 hours, presumably from gastrointestinal tract absorption.⁷⁵⁷ When the cyclopentolate drops were separated by 5 minutes, the peak concentration, 8.3 ± 0.5 ng/mL, was reached 10 ± 5 minutes after the second drop¹⁸⁹; the second peak occurred at 45 to 60 minutes. The mean half-life in plasma of both atropine and cyclopentolate is just under 2 hours.⁷⁵⁸ Two 40-μL drops of 0.5% tropicamide instilled in one eye of women whose mean age was 70.4 ± 9.3 years produced a peak concentration of 2.8 ± 1.7 ng/mL in 5 minutes.⁷⁵⁹ By 5 hours, the plasma level was less than 0.25 ng/mL.

Death occurred in a 3-year-old white girl with a corneal perforation who was given six drops of 1% atropine topically, 0.1 mg atropine subcutaneously, and, at the conclusion of surgery, 1.5 g of 1% atropine ointment on the lids.⁷⁶⁰ The patient died within 24 hours of the first application of atropine. A 3-year-old white retarded girl developed a fever, convulsed, and died after receiving only four drops of 1% atropine; no postmortem examination was performed. Scopolamine,⁷⁵⁴ homatropine,⁷⁶¹ and cyclopentolate^762–765 have produced toxicity. The central nervous system manifestations include cerebellar signs, ataxia, dysarthria, and inappropriate behavior such as hallucinations, incoherent speech, and restlessness. Patients with prior histories of convulsions may develop seizures more readily.⁷⁶⁶ Dry, flushed skin and fever are often but not always present.^761,767 Toxic signs may occur within a few minutes of administration.⁷⁶³ Recovery occurs within 24 hours. Two studies stand out as being especially well documented. In one, an 8-year-old black girl developed toxicity after two drops of 1% tropicamide and four drops of 1% cyclopentolate.⁷⁶⁷ The child was subsequently readmitted and given 10 drops of 1% tropicamide within 30 minutes. Toxicity did not develop. Five days later, she was challenged with six drops of 1% cyclopentolate within a 15-minute period. Toxicity, including hallucinations, returned. The other report was by Binkhorst and co-workers.⁷⁶⁸ It was a masked prospective study of drug toxicity. Thirty-five children had psychiatric evaluations before and after receiving four drops bilaterally of 1% cyclopentolate, 2% cyclopentolate, 1% homatropine, or 1% hydroxyamphetamine. Each of the four sets of drops was instilled 10 minutes apart. Only the 2% cyclopentolate solution evoked significant abnormal behavior.

Newborns are especially susceptible to systemic intoxication. Reports have associated increased abdominal distention and necrotizing enterocolitis with the use of muscarinic antagonist eyedrops for ophthalmic examination.^765,769 Isenberg and colleagues⁷⁷⁰ found that 0.25% cyclopentolate had no significant effect on gastric function but that 0.5% cyclopentolate did. Interestingly, these workers reported that, as would be expected, 0.5% cyclopentolate reduced gastric volume output, whereas Hermansen and Sullivan⁷⁶⁹ reported that 0.5% cyclopentolate was associated with large gastric aspirates.

Systemic toxicity from muscarinic antagonists has been treated with direct and indirect muscarinic agonists. Pilocarpine, 6 mg subcutaneously, has caused death in a 3-year-old child.⁷⁶⁰ Forrer and Miller⁷⁷¹ reversed the delirium and coma that followed the ingestion of 200 mg or more of atropine by injecting 1 to 4 mg physostigmine. The reversal was temporary, and after 30 to 60 minutes additional physostigmine had to be given. Duvoisin and Katz⁷⁷² and Young and colleagues⁷⁵⁴ suggested using an initial subcutaneous dose of 0.25 mg physostigmine in children and 1 to 2 mg physostigmine in adults. This initial dose would be supplemented as needed every 15 minutes by doses of similar magnitude.

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