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Introduction
Muscarinic receptors initiate many important
physiological actions of acetylcholine, a major neurotransmitter in the
nervous system. Molecular cloning and pharmacological
studies have revealed 5 distinct muscarinic receptors referred to
as M1_M5[1]. Central muscarinic receptors are involved in
cognitive, behavioral, sensory, motor, and autonomic
functions. The peripheral actions mediated by muscarinic
receptors include a reduction of heart rate, stimulation of
glandular secretion, and smooth muscle
contraction[2]. Reduced or increased signaling through muscarinic receptors
has been implicated in the pathophysiology of several major
diseases[3]. Chemical compounds
that influence central and peripheral muscarinic function have long been the focus of
intensive research, since the development of new highly
potent muscarinic receptor agonists could provide novel
therapeutic agents useful, for example, in the treatment of
glaucoma, pain, and Alzheimer's disease.
The cholinergics were the first class of agents used for
the treatment of glaucoma, a disease characterized by the
degeneration of optic nerve axons and death of retinal
ganglion cells, and frequently associated with high intraocular
pressure[4]. It was considered that muscarinic cholinergics
improved the trabecular outflow of aqueous fluid by
opening trabecular meshwork action. However, it now appears
that they also limit the production of aqueous humor and
have protective and trophic effects on retinal ganglion
cells[5_7]. As a consequence, there has recently been renewed interest
in the application of muscarinic-based therapies in the
treatment of glaucoma[8].
Baogongteng A (6β-acetoxy-2β-hydroxy-nortropane), a
nortropane alkaloid from the Chinese herb Erycibe
obtusifolia Benth, first isolated by our
laboratory[9,10], has been demonstrated to posses potent agonistic activity on muscarinic
receptors and was developed into an antiglaucoma agent in
China. Clinical trials demonstrated that the therapeutic
efficacy was similar to pilocarpine in the treatment of primary
glaucoma[11_13]. However, the low amount of baogongteng A
available from the herb limits its extensive clinical application.
Thus, great efforts has been taken in the
synthesis of baogongteng A and its analogs by our laboratory and
others in recent decades[14_19]. Satropane (racemic
3α-paramethyl-benzenesulfonyloxy-6β-acetoxy-tropane), a
novel tropane analog synthesized in our laboratory, was
shown to be a promising candidate as a new antiglaucoma
agent in our previous preclinical studies.
Data that the agonist (and the antagonist) binding sites
of muscarinic receptors are asymmetrical, and hence
generally capable of distinguishing between optical isomers of
chiral ligands have been
accumulated[20_22]. It is reasonable to suppose that enantiomers of satropane may behave as
different compounds on interaction with muscarinic
receptors, and the stereospecific interactions of racemic
satropane at recognition sites in muscarinic receptors may
result in differences in both biological and toxicological
effects. Recently, we resolved satropane into a pair of
enantiomers, S(_)satropane (lesatropane) and
R(+)satropane (see Figure 1 for structural
formula)[15].
In order to study the stereoselectivity of satropane on
iris muscarinic receptor activation and intraocular
hypo-tension, we investigated the pharmacological
characteristics of these 2 chiral compounds by comparing their effects
on muscarinic receptors in rabbit eyes in
vitro and in vivo. The binding characteristics, contractile responses of
isolated rabbit iris muscle, miotic response of the conscious
rabbit, and intraocular hypotension of the enantiomers of
satropane were investigated. The results revealed that the
agonistic and hypotensive properties of satropane on rabbit
eyes were stereoselective, with the S(_)isomer being its
active form.
Materials and methods
Drugs R,S(±)satropane and its enantiomers were
synthesized in our department, as previously
described[15]. The enantiomeric excess of
S(_)satropane and R(+)satropane was 98.05% and 100.00%, respectively, analyzed on a chiral HPLC
column (Chiralpack AD). Carbachol, pilocarpine, atropine,
pirenzepine, gallamine,
4-diphenylacetoxy-N-methylpiperi-dine (4-DAMP), and
tris-(hydroxymethyl) amino methane (Tris) were obtained from Sigma (St Louis, MO, USA), and
[3H]quinuclydinyl benzilate
([3H]-QNB; spec. act. 43 Ci/mmol) was from Amersham (Buckinghamshire, England).
Animal and tissue preparation New Zealand
rabbits (2.5±0.5 kg, Certificate No SCXK 2002-0006) were
treated in accordance with the University Guide for the Care
and Use of Laboratory Animals. In the in
vitro studies, the eyes were immediately enucleated and the iris smooth
muscle was excised for the binding assay and isolated iris
contraction assay after the animals were killed by injecting
atmospheric air into the marginal ear vein.
Radioligand-receptor binding assay The iris muscle was
minced with scissors in ice-cold 50 mmol/L Tris buffer (pH 7.4).
The tissue was then homogenized in 1g of 20 mL
(w/v) volume ice-cold 0.32 mol/L sucrose in Tris buffer using a
Waring blender (IKA, Staufen, Germany) and further disrupted
with an Ultraturrax tissuemizer (IKA, Staufen, Germany).
The crude homogenate was centrifuged for 10 min at
1000×g and the resulting supernatant was centrifuged for 60
min at 20 000×g. The pellet was resuspended in Tris buffer
as a crude membrane fraction. All the procedures were
performed at 4 °C. In the saturation binding assay, the
membranes (0.1 mg protein) were incubated vibrantly at 32 °C for
30 min with 0.05_1.1 nmol/L [3H]QNB in a total volume of 0.4
mL. The reaction was terminated by rapid filtration through
GF/C glass fiber filters and washed 3 times with ice-cold Tris
buffer. The protein concentration was determined with the
micro BCA kit (Pierce, Rockford, IL, USA), using bovine
serum albumin as the standard. For the competition binding
assays, iris muscle membranes (0.1 mg protein) were incubated
with 0.4 nmol/L [3H]-QNB at 32 °C for 60 min with increasing
concentrations of the agonists carbachol, pilocarpine,
S(_)satropane, and R(+)satropane, respectively, in a total
volume of 0.4 mL. All the dilutions for the agonists were made in
Tris buffer. Non-specific binding was measured in the
presence of 10 µmol/L atropine sulfate and accounted for
5%_12% of the total binding. Assays were performed in
duplicate.
Isolated iris contraction assay The freshly prepared
iris muscle was mounted in 10 mL organ chambers
containing modified Krebs-Henseleit solution containing (in
mmol/L): 118 NaCl, 4.7 KCl, 2.5 CaCl2, 25
NaHCO3, 1.2 MgSO4, 1.2
KH2PO4, 11.0 glucose, and 0.5
EDTA·Na2. The bath was continuously aerated with
aO2:CO2 mixture (95%:5%) and kept at a constant temperature of 37 °C. The
preparation was connected vertically to a force-displacement
transducer under a resting tension of 500 mg.
Preparations were allowed to equilibrate for at least 60 min before
the drug addition, during which the buffer solution was
refreshed every 15 min. Isometric contractions were
recorded using a PowerLab/8sp life analysis system
(AD-Instruments, Australia). In order to confirm the viability of
the tissue, preparation was exposed to a high potassium
concentration (60 mmol/L KCl) following the stabilization
period. After washout replacement with normal medium
and return to the original baseline, the cumulative
concentration_response curves were obtained for carbachol,
pilocarpine, R,S(±)-satropane,
S(_)satropane, and R(+)satropane, respectively. The concentration was increased
as soon as a stable response to the previous concentration
appeared. The contractile responses of the iris muscle to
each dose of the muscarinic agonists are expressed as
percentages of that elicited by 100 μmol/L carbachol. In our
pilot study, carbachol at this concentration could induce
the maximum contraction. No significant desensitization
was observed for at least 2 consecutive
concentration_response curves for the compounds. Accordingly, no more
than 2 complete curves were recorded for each tissue. In
order to investigate whether the active isomer of satropane
produced iris contraction through the muscarinic receptor
and to determine the subtype of the receptor mediating the
effects, muscarinic receptor selective antagonist atropine
and muscarinic receptor subtype selective antagonists
piren-zepine (for M1), gallamine (for M2), and 4-DAMP (for
M3) were used[23,24]. The antagonists were added to the
preparations 20 min before the administration of the agonists.
Different sets of preparations (1, 10, and 100 μmol/L) of the
inactive isomer of satropane were pre-incubated to
determine if the isomer without agonistic effect elicits an
antagonist profile. All concentrations of drugs are expressed as the
final concentration in the organ bath.
Pupil diameter and induced ocular hypertension
measurements The compounds were dissolved in physiological
saline (9 g/L sodium chloride solution) and each eye was
instilled 100 µL eye-drop solution. In each following test, 6
different dosing studies (20 eyes of 10 rabbits per group)
were evaluated in random order and included physiological
saline, pilocarpine (2%), S(_)satropane (0.015%, 0.03%,
and 0.06%) and R(+)satropane (0.12%;
w/v).
For measuring the pupil diameter, conscious rabbits were
placed in restraint boxes to which they had been habituated,
with unrestricted head or eye movements. The pupil
diameter (in mm) was measured with a Castroviejo caliper
under normal room lighting and readings were taken before
(taken as 0 min) and at 15, 30, 60, 120, 180, and 240 min
after the application of compounds.
Water loading-induced ocular hypertension and
methylcellulose-induced ocular hypertension were assessed as
described by Konno et al with slight
modifications[25_27]. Briefly, the orogastric administration of 100 mL/kg (37 °C) of
distilled water into rabbits was for acute ocular hypertension.
For inducing methylcellulose-induced ocular hypertension,
a single injection of 2% methylcellulose (in sterile saline
solution) with a 30 gauge needle was introduced into the
posterior ocular chamber of the eyes of the rabbits
anesthetized by an intravenous injection of 30 mg/kg sodium
pentobarbital and a topical administration of 1% tetracaine.
The intraocular pressure of both eyes was measured
using Schiotz tonometers (Suzhou Medical Instruments,
Suzhou, China) immediately before (taken as 0 min or h) and
at 15, 30, 60, 120, and 180 min after the administration of
distilled water in water loading-induced ocular hypertension
test or at 0.5, 2, 4, 6, and 24 h after the injection of
methylcellulose in the methylcellulose-induced hypertension test. The
compounds tested were instilled immediately after the
administration of distilled water or methylcellulose.
Statistics and data analysis In the binding tests,
non-linear curve fitting by GraphPad PRISM 4.0 (San Diego, CA,
USA) was used to generate affinity
(Kd) and capacity
(Bmax) values for [3H]-QNB and the competition parameters. The
apparent dissociation constants
(Ki) were calculated from
IC50 values according to the Cheng-Prusoff equation, and
the values were expressed as pKi (_lg
Ki). The only variables constrained in the analysis were those that were
experimentally determined, namely, the dissociation constant for
[3H]-QNB and the non-specific binding of
[3H]-QNB. For the iris contraction assay,
EC50 (concentration of an agonist produce 50% of the maximal response of the agonist) values
were calculated by means of non-linear curve fitting of
sigmoidal dose-response logistic transformation using PRISM
4.0. The negative logarithm to base 10 of the equilibrium
dissociation constant pKb values for the antagonists were
determined by Schild analysis. Data were expressed as the
mean±SD of 3 independent experiments unless otherwise
stated.
The statistically significant differences were determined
by Student's t-test or by ANOVA, as appropriate.
Differences were considered statistically significant if
P<0.05.
Results
Enantiomers of satropane binding to iris
muscle The binding of [3H]-QNB was saturable. The dissociation
equilibrium constant (Kd) and receptor density
(Bmax) were determined to be 0.22±0.09 nmol/L and 1.25±0.08
pmol·mg_1 protein (n=3), respectively.
Displacement of [3H]-QNB binding was performed using
carbachol, pilocarpine, S(_)satropane and
R(+)satropane. [3H]-QNB binding on the muscarinic receptor was inhibited
by the compounds in a concentration-dependent manner
(Figure 2). The maximum inhibition and the
pKi values of the compounds against
[3H]-QNB binding are summarized in Table 1. With the exception of
R(+)satropane, all the compounds were completely against the labeled ligand binding
with muscarinic receptors in the iris muscle.
Enantiomers of satropane on the contraction of isolated
rabbit iris muscle The cumulative addition of carbachol,
pilocarpine, and S(_)satropane to the isolated iris muscle
produced a concentration-dependent contractile response
(Figure 3). However, R(+)satropane did not induce any
contractile response up to the concentration of 300
μmol/L. The parameters of the dose_response are shown in Table 2.
Carbachol and S(_)satropane were the most potent, and
pilocarpine was least potent with approximate one-ninth potency
of carbachol and S(_)satropane. The efficacy of carbachol,
pilocarpine, and S(_)satropane varied. Carbachol was most
efficacious, inducing the maximum contraction of the iris
muscle (0.41±0.11 g). Pilocarpine was least efficacious,
eliciting less than 23% of the maximal response to
carbachol (P<0.01). S(_)satro-pane stimulated the contraction of the
iris muscle, with the maximum contraction near that of
carbachol (P>0.05) and greater than that of pilocarpine
(P<0.01).
Effect of S(_)satropane on the contraction of the isolated
rabbit iris muscle under the pre-incubation of the
muscarinic receptor antagonists or
R(+)satropane Pre-incubation of the preparations with the various concentrations of
muscarinic receptor selective antagonist atropine or the
M3 subtype selective antagonist 4-DAMP made the dose-response
curves of S(_)satropane shift rightward in a parallel manner.
The pKb values of atropine and 4-DAMP were 9.12±0.09 and
9.10±0.08, respectively. The M1 subtype selective antagonist
pirenzepine up to 100 nmol/L and M2 subtype selective
antagonist gallamine up to 1 μmol/L failed to shift the
S(_)satropane concentration_response curve (Figure 4). Pre-incubation
with R(+)satropane up to 100 μmol/L had no effect on the iris
contraction induced by carbachol (data not shown).
Effect of enantiomers of satropane on conscious rabbit
pupil diameter The basal pupil size was 6.01±0.03 mm before
drug treatment. 0.12% R(+)satropane failed to induce miosis.
Pilocarpine (2%) and S(_)satropane (0.015%, 0.03%,
and 0.06%) significantly decreased the pupil diameter after the
topical administration until 120 min, with the maximal effect
at 15 or 30 min (Figure 5). At 15 min, pilocarpine decreased
the pupil diameter to 4.21±0.16 mm, and at 30 min, it decreased
to 4.32±0.09 mm. The significance of miosis produced by
S(_)satropane was concentration dependent, with 0.06%
S(_)satropane eliciting the maximum effect. The pupil diameter
was lowered to 3.51±0.16 mm at 30 min after instillation with
0.06% S(_)satropane compared to 6.12±0.15 mm after
instillation with physiological saline. S(_)satropane was more
potent in inducing miosis than pilocarpine.
Effect of enantiomers of satropane on water
loading-induced and methylcellulose-induced ocular hypertension
The basal intraocular pressure was 2.65±0.08 kPa
(n=20) before drug treatment. Water loading caused a rapid increase in
intraocular pressure to 4.70±0.17 kPa at 15 min, 4.54±0.15 kPa
at 30 min, and decreased near to the baseline at 180
min. Eye dropping of pilocarpine (2%) and
S(_)satropane (0.03% and 0.06%) significantly suppressed water loading-induced ocular
hypertension from 15 min after the administration of water
until 120 min. S(_)satropane was more potent in reducing
intraocular pressure than pilocarpine (Figure 6A).
The injection of methylcellulose into the posterior
chamber of the rabbit eye produced an elevation in intraocular
pressure, which reached its maximum of 6.75±0.36 kPa 2 h
after the injection and decreased until it was stabilized at a
level of approximately 3.53_4.54 kPa for 20 h (Figure 6B).
Pilocarpine (2%) and S(_)satropane (0.015%, 0.03%, and
0.06%) significantly suppressed methylcellulose-induced
ocular hypertension throughout the whole duration tested.
S(_)satropane was more potent in reducing intraocular pressure
than pilocarpine. R(+)satropane neither suppressed water
loading-induced nor methylcellulose-induced ocular
hypertension.
Discussion
Muscarinic receptors play key roles in the central and
peripheral nervous system. Molecular cloning and
pharmacological studies have revealed 5 distinct muscarinic
receptors referred to as M1_M5[24]. The iris-ciliary body contains
parasympathetic innervation and contributes to the
regulation of intraocular pressure and pupil
diameter[28]. The main effects on iris contractility and outflow facilitation are
mediated by muscarinic
stimulation[29,30]. A number of techniques
have revealed that it is the M3 subtype that appears to be
the most abundant muscarinic receptor expressed in the iris
of humans and other mammals. The contraction of the iris
by muscarinic agonists is also primary mediated by the M3
receptor[31_34].
In our study, the enantiomers of satropane inhibited
[3H]-QNB binding on the muscarinic receptor in a
concentration-dependent manner. S(_)satropane completely competed
against the labeled ligand as carbachol and pilocarpine did,
whereas R(+)satropane did not. It is likely that
R(+)satropane has very weak binding affinity with muscarinic receptors in
the iris muscle. R(+)satropane did not induce miosis or
suppress hypertensive intraocular pressure induced by water
loading or by methylcellulose posterior ocular chamber
injection, but S(_)satropane induced these effects at a much
lower concentration than pilocarpine. Moreover, in the
isolated iris assay, R,S(±)satropane and
S(_)satropane produced a potent contractile response, while
R(+)satropane did not. Pre-incubation with
R(+)satropane had no effect on the iris contraction induced by carbachol,
indicating that R(+)satropane did not behave like an
antagonist. The potency and the efficacy of
S(_)satropane was similar to carbachol. In this way,
S(_)satropane behaved like a fully or highly efficacious partial agonist, whereas
R(+)satropane elicited neither agonistic nor antagonistic
activity in inducing the contraction of the iris muscle both
in vitro and in vivo. The contractile responses of
S(_)satropane on the isolated iris muscle were blocked by muscarinic
receptor antagonist atropine and M3 subtype selective
antagonist 4-DAMP, but hardly by M1 subtype selective
antagonist pirenzepine and M2 subtype selective allosteric
antagonist gallamine. The effects of S(_)satropane is
mediated by a M3-like receptor subtype in the iris muscle.
Besides lesatropane, many other 6β-acetoxy(nor)tropane
analogs have been demonstrated to elicit potent agonist
activity at muscarinic
receptors[18,35_38]. However, their
tropane counterparts, such as atropine, scopolamine, anisodine,
and anisodamine, are generally known potent muscarinic
receptor antagonists. The tropane alkaloids appear as
useful tools to study the physiological roles and provide an
interesting starting point for the analysis of
structure_activity relationships at muscarinic receptors. The significance
of molecular chirality is widely recognized in life
sciences[39_41]. Although the use of chiral drugs predates modern medicine,
it is only since the 1980s has there been a significant
increase in the development of chiral pharmaceutical drugs,
primarily due to the recognition that enantiomers often have
different bioactivity and metabolic fates. Additional isomers
in a compound are no longer considered "silent passengers",
but potential contaminants (so-called isomeric
ballast)[42]. The enantiomers of satropane behave as different
compounds on interaction with muscarinic receptors, and the
stereospecific interactions of racemic satropane at
recognition sites in muscarinic receptors may result in differences in
both biological and toxicological effects. In the present study,
S(_)satropane rather than R(+)satropane elicited agonistic
activity on muscarinic receptors and suppressed
hypertensive intraocular pressure. It is possible
that R(+)satropane without pharmacodynamic effects could behave as a
potential contaminant with the administration of racemic satropane
to patients. The chemical and pharmacodynamic separation
of the opposite configurations of satropane is likely to
assist in further research and discovery of this kind of
muscarinic receptor agonist. The exploration and development of
single isomer drugs may bring significant advances in
treatment options.
In conclusion, satropane exhibits significant agonistic
effect on the iris muscarinic receptor, and the agonistic and
hypotensive properties of satropane on rabbit eyes are
stereoselective with the S(_)isomer lesatropane being its
active form.
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