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Introduction
In order to develop more potent drugs for muscarinic
intervention in disorders of the central and peripheral
nervous system, a series of heterocyclic compounds including
esters, alkanes, and ether derivatives were designed and
synthesized in our institute. One of the compounds, CPG,
has been developed as a novel anti-motion-sickness drug
with higher efficacy and lower central inhibitory side effects
than diphenidol HCl and scopolamine HBr, and is used
clinically[1-3]. (±)DMCPG, which has one chiral center and two
enantiomers [R(-) and S(+)DMCPG] was the active
dominant N-demethyl metabolite of CPG. We believe that this is
an important way of looking for active compounds from the
metabolites to discover new medicines. We synthesized
(±)DMCPG and its two enantiomers (Figure 1). To avoid
adverse effects and to optimize the therapeutic value of
enantiomeric drugs, it is necessary to establish the effectiveness
of isomers of the chiral drug, and to detect the presence of
the enantiomer with lower therapeutic activity and
undesirable adverse effects. For this purpose, we investigated the
binding characteristics of these new chiral compounds by
using a radioligand receptor-binding assay with muscarinic
receptors from rat brain, and compared its pharmacological
effects on muscarinic receptors by means of in
vitro and in vivo assays.
Materials and methods
AnimalsThe experiments were carried out in accordance
with the Guide for the Care and Use of Laboratory Animals,
from the National Research Council (1996), under the
approved protocols. Animals used in the present study were
as follows: Kunming species mice weighing 18-22 g [grade
II, certificate No scxk (Army) 2002-001], provided by our
animal center; male and female Wistar rats weighing 180-220 g
[grade II, certificate No scxk (Jing) 2002-0003], and male guinea
pigs weighing 200-300 g [grade II, certificate
No scxk (Jing) 2002-0003] purchased from Weitonglihua Co (Beijing, China).
Compounds 3H-quinuclindinyl benzilate
([3H]QNB; 43.3 Ci/mmol) was purchased from Amersham (Uppsala, Sweden;
TRK604). (±)DMCPG and its isomers were synthesized at
our institute. The (±)DMCPG comprised equal proportions
of R(-) and S (+)DMCPG. Atropine, pentobarbital,
oxotremorine and carbachol were purchased from Sigma (St Louis,
MO, USA).
Binding assays on rat cerebral cortex homogenate
Male or female Wistar rats were killed by decapitation. The
cerebral cortex was immediately removed and processed as
described by Yamamura and Snyder[4].The protein
concentration was determined by using the method of Lowry
et al[5].
The samples of homogenate (each containing 50 mg of
protein) were incubated for 30 min at 37 ºC in 0.5 mL of assay
buffer containing 6 nmol/L [3H]QNB and various
concentrations of drugs. In saturation binding assays, the
homogenate was incubated as indicated above, in the presence of
[3H]QNB (0.25-20 nmol/L). Non-specific binding was
defined as binding in presence of atropine (1 µmol/L). Each
sample was filtered through GF/C glass fibers with vacuum.
The filters were rinsed 3 times with 3 mL cold buffer, and
placed in scintillation vials containing 3 mL of scintillation
fluid. Radioactivity trapped on the filters was determined by
liquid scintillation spectrometry at approximately 40%-50%
efficiency.
Carbachol-induced contraction Male guinea pigs were
killed by cervical dislocation. The organs required were set
up rapidly under 1 g of tension in 20 mL organ baths
containing physiological salt solution (PSS), which was kept at
37 ºC and aerated with 5% CO2 and 95%
O2. Two-centimeter-long portions of terminal ileum were taken at about 5 cm from
the ileum-cecum junction and mounted in PSS at 37 ºC. The
composition of PSS was as follows (mmol/L): NaCl 118,
NaHCO3 23.8, KCl 4.7,
MgSO4·7H2O 1.18,
KH2PO4 1.18,
CaCl2 2.52, glucose 11.7. Tension changes were recorded
isotonically. Tissues were equilibrated for 90 min before the experiments
were conducted.
The carbachol-induced ileum contraction was obtained
by incubation with carbachol (10-5 mol/L) until the
concentration had reached a plateau. Then, the ileum strips were
washed several times with PSS until the tension of the strips
relaxed to the baseline level. Following washing, the strips
were incubated with different concentrations of R(-), S(+),
or (±)DMCPG for 10-15 min. After incubation, the maximum
contraction induced by carbachol (10-5 mol/L) was observed
again in the presence of increased concentrations of
different antagonists. The IC50 values for the carbachol-induced
contractions in the presence of the antagonists were
obtained to assess the pharmacological potency of (±)DMCPG
and its optical isomers.
Effect on sub-threshold hypnotic dose of sodium
pentobarbital induced-sleep Four dosage groups were used for
each drug and each group consisted of 10 mice of each sex.
Mice were pretreated with (±)DMCPG and its optical
isomers intraperitoneally (ip), then after 15 min, a sub-threshold
hypnotic dose of sodium pentobarbital (30 mg/kg) was given
ip. The loss of righting reflex was used as a measure of the
central inhibitory effect of drugs. The
ED50 values of these three drugs were estimated to compare the central inhibitory
effect of the indicated agents.
Inhibition of oxotremorine-induced salivation
Kunming
mice were assigned randomly into 4 groups for each drug.
Each group consisted of 10 mice of each sex. (±)DMCPG
and its optical isomers were administered ip 15 min prior to
oxotremorine (3 mg/kg) being injected subcutaneously (sc).
ED50 values were calculated to evaluate the anti-secretive
potencies of the compounds used.
Data analysis and statistics
Binding assays The IC50 values were obtained from at
least three separate experiments performed in triplicate with
6-8 concentrations of drugs. Data were analyzed by
curvilinear regression using the program ORIGIN 6.0. The
inhibition constants (Ki) were calculated using the Cheng-Prusoff
equation[6],
Ki=IC50/(1+L/K
d), where L and Kd are the
concentration and the equilibrium dissociation constant of
[3H]QNB, respectively.
Functional assays In the carbachol-induced
contraction experiments, the maximum contractile response
(Emax) was obtained from the maximum stress, and the
IC50 value was calculated from a semi-logarithmic plot of the
percentage of the maximum response versus drug concentration.
Data were computer analyzed by curvilinear regression
using the program ORIGIN 6.0. Statistical analyses for
comparisons among groups were performed using analysis of
variance (ANOVA). P<0.05 was considered statistically
significant. To evaluate the effect of (±)DMCPG and its
optical isomers on anti-salivation induced by oxotremorine and
sleeping induced by a sub-threshold hypnotic dose of
sodium pentobarbital, ED50±95% CL (confidence limit) values
were calculated and compared by weighted probit analysis.
Data are shown as mean±SD.
Results
Competitive binding of (±)DMCPG and its optical
isomers to rat central muscarinic acetylcholine receptors
The Kd value for
[3H]QNB binding to receptors was 6.66±0.95
nmol/L. The Bmax was 760±92 fmol/mg. The competitive
binding potency of R(-)DMCPG for [3H]QNB corresponded to a
Ki value of 763.75±7.31 nmol/L
(n=4). An average Hill coefficient
(nH) was 1.17±0.15. The affinity of R(-)DMCPG at
central muscarinic acetylcholine receptors was higher than that
of (±)DMCPG (Ki=3180±263 nmol/L,
nH=0.42) and S(+) DMCPG
(Ki=1699±260 nmol/L,
nH=1.26). The isomer with
R(-) configuration was more potent than the isomer with
S(+) configuration and (±)DMCPG. The competition
profiles of R(-)DMCPG and S(+)DMCPG to rat cortex
muscarinic receptors were steep and adequately described by a
one-site model, nH>1, which exhibited positive cooperative
effects at muscarinic receptors. However, for (±)DMCPG,nH<1, which exhibited negative cooperative effects at
muscarinic receptors, which is not consistent with a one-site
binding model (Figure 2).
Effect of (±) DMCPG and its optical isomers on
carbachol-induced contraction Carbachol
(10-5 nmol/L) caused contractions in guinea pig ileum. The
Emax values for the carbachol-induced contractions were 2.90±0.17 g
(n=30).
(±)DMCPG and its optical isomers
(10-9-10-4 mol/L)
significantly suppressed the carbachol-induced contractions
(P<0.05; Figure 3). The IC50 values of (±) DMCPG ,
R(-)DMCPG and S(+)DMCPG were
7.78×10-9,
1.88×10-7, and 1.03×10-7
mol/L, respectively. The results revealed that
(±)DMCPG was more potent in the anti-contraction of smooth
muscle induced by carbachol than the other two
configurations (P<0.05).
Potentiation of the effect of a sub-threshold hypnotic dose
of sodium pentobarbital Pentobarbital (ip, 30 mg/kg) alone
did not cause sleep in mice (n=50). However, after
pretreatment with R(-)DMCPG (1.68-4.89 mg/kg) and S(+)DMCPG
(10-29.15 mg/kg) at 15 min intervals, sedation effects induced
by a sub-threshold hypnotic dose of sodium pentobarbital
were enhanced in a dose-dependent manner (Table 1). The
ED50 values ±95% confidence limits of R(-) and S(+)DMCPG
were 2.53±0.37 and 18.65±4.03 mg/kg. Of the 10 mice
pre-administered with (±)DMCPG at the highest dose (29.15
mg/kg), only 2 mice lost their righting reflex, which indicates
that (±)DMCPG has a weak central depressant action. The
order of central inhibition effects was R(-)DMCPG>
S(+)DMCPG>(±)DMCPG.
Inhibition of oxotremorine-induced salivation
Oxotremorine (sc, 3 mg/kg) induced obvious salivation in mice
(n=50), whereas (±)DMCPG and its optical isomers produced
anti-salivation effects in a dose-dependent manner when mice
were pretreated with these compounds. The
ED50±95% CL values
for (±)DMCPG and the R(-), S (+) configurations were
2.88±0.35, 0.44±0.03, and 5.05±0.33 mg/kg, respectively, which
indicates that the order of potency for inhibiting glandular
secretion is R(-)DMCPG>(±)DMCPG>S(+)DMCPG
(Table 2).
Discussion
(±)DMCPG is a derivative of its parent compound CPG,
and there is one chiral carbonic atom in the molecular
structure of (±)DMCPG, causing the R(-) and S(+) enantiomers.
In this study, we compared the pharmacological activities of
these enantiomers, to further investigate the relationships
between anti-muscarinic activity and muscarinic receptors.
Interestingly, for muscarinic acetylcholinic receptors,
(±)DMCPG and its optical isomers did not have the same
potency trends in the tests.
In the present investigation, the pharmacological
activities of (±)DMCPG and its isomers were subjected to
comparative radiobinding and functional assays. In the
competitive binding assay, R(-)DMCPG was 4- and 2-fold more
potent than its racemate and the S(+) configuration in
inhibiting the binding of [3H]QNB. These results demonstrate
that there was receptor stereo selective action between the
muscarinic receptor and R (-)DMCPG. In this case of
receptor binding, R(-)DMCPG is the eutomer. The
R(-) and S(+) configurations showed positive cooperation
(nH>1) but
(±)DMCPG had a negatively cooperative
(nH=0.42) relationship with the muscarinic receptor. Low Hill numbers are
most often attributed to recognition by the antagonist of
more than one receptor site or receptor conformation, or to
an interaction of the antagonist with a second binding site
on the receptor molecule causing a negative cooperative
effect for the first site[7-9]. There is a second ligand-binding
site on muscarinic receptors. A wide array of compounds is
capable of modulating the binding of classical ligands to all
five muscarinic subtypes[10-12]. Allosteric modulation of
muscarinic receptors has been much
investigated[13,14]. Proška and Tuèek (1994) proposed that the binding site for
modulators such as alcuronium, gallamine, and related compounds
is located near the binding site for classical ligands, but more
superficially[15]. Our results imply that (±)DMCPG may act at
the second binding site with an allosteric mechanism to
muscarinic receptors. (±)DMCPG is composed of the
R(-) and
S(+) configurations, and R(-) and S(+)DMCPG showed
marked central inhibitory effects, but (±)DMCPG had only a
weak effect, even at a higher dose. Inversely, the ability of
(±)DMCPG to inhibit the contraction of guinea pig ileum
induced by carbachol was approximately 10 and 100 times
more potent than that of R(-) and S(+)DMCPG. (±)DMCPG
was the lowest one bound to the muscarinic receptor, and
we can also deduce that the enantiomers must interact with
each other to affect its binding characteristics and
bioactivities. The allosteric mechanism of these chiral drugs
needs to be further explored in subsequent experiments.
However, our experiments showed that pharmacological
differences exist between the optical isomers. The R(-)
configuration was more potent at binding receptors and
inhibiting glandular secretion, but had moderate effects on the
contraction of smooth muscle. The pharmacological differences
may be due to the distribution of different subtypes in
different tissues. Muscarinic acetylcholine receptors (mAChR)
include five subtypes of receptors
(M1-M5). The selective action on the salivary gland of R(-)DMCPG may be related
to its subtype-selective effects. Individual members of the
mAChR are expressed in a complex overlapping fashion in
most tissues and cell types[16]. The
M1, M2, and M4
subtypes of the mAchR are the predominant receptors in the
central nervous system[17]. The
M1 and M3 subtypes are the major muscarinic acetylcholine receptors in the salivary
glands and M3 is thought to be more
abundant[18,19]. Guinea pig ileum smooth muscle is enriched with muscarinic
receptors, the majority of which are of the
M2 subtype, and the remaining minority are of the
M3 subtype[20,21]. In our experiments, we comparatively studied the anti-muscarinic
pharmacological profiles of these compounds to see whether
there is any correlation between pharmacological activity
and muscarinic subtype selectivity. Fulfilling our
expectations, of these three chiral drugs, (±)DMCPG had the
greatest ability to inhibit smooth muscle contraction as a
muscarinic receptor antagonist, whereas R(-)DMCPG had a
moderate effect on smooth muscle contraction, but had the
greatest anti-salivary effect and enhancement of sedation
effects caused by sub-threshold hypnotic doses of sodium
pentobarbital. S(+)DMCPG was less potent in all
experimental models. Our results imply that there are
subtype-selective mechanisms that correspond to the different
pharmacological actions of the compounds. Therefore, further
studies are necessary to resolve the underlying actions of
(±)DMCPG and its enantiomers with respect to muscarinic
subtype receptors.
In conclusion, the present work demonstrated that that
there are receptor stereo selective actions between
muscarinic receptors and the R(-) configuration of DMCPG.
R(-) DMCPG acted as a eutomer relative to its S(+) configuration
in the racemate. These differences must be related to
subtype selectivity and allosteric mechanisms.
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