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Full paper
RQ-00201894: A motilin receptor agonist causing long-lasting
facilitation of human gastric cholinergically-mediated contractions
John Broad
a
, Nobuyuki Takahashi
c
, Masaomi Tajimi
c
, Masaki Sudo
c
, Adam G
oralczyk
b
,
Umesh Parampalli
b
, Kesava Mannur
b
, Toshinori Yamamoto
c
, Gareth J. Sanger
a
,
*
a
Blizard Institute (National Centre for Bowel Research and Surgical Innovation), Barts and the London School of Medicine and Dentistry, 2 Newark Street,
London, E1 2AT, UK
b
Bariatric Surgery Department, Homerton University Hospital, Homerton Row, London, E9 6SR, UK
c
RaQualia Pharma Inc., Department of Pharmacology, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya,
464-8601, Japan
article info
Article history:
Received 24 June 2015
Received in revised form
5 October 2015
Accepted 11 November 2015
Available online xxx
Keywords:
Motilin
RQ-00201894
Human stomach
Cholinergic activity
Gastroparesis
abstract
The aim was to characterise RQ-00201894, a novel non-macrolide motilin agonist, using human re-
combinant receptors and then investigate its ability to facilitate cholinergic activity in human stomach. A
reporter gene assay assessed motilin receptor function. Selectivity of action was determined using a
panel of different receptors, ion channels, transporters and enzymes. Cholinergically-mediated muscle
contractions were evoked by electrical field stimulation (EFS) of human gastric antrum. The results
showed that RQ-00201894, motilin and erythromycin acted as full motilin receptor agonists (EC
50
: 0.20,
0.11, 69 nM, respectively). In this function, RQ-00201894 had >90-fold selectivity of action over its ability
to activate the human ghrelin receptor (EC
50
19 nM) and greater selectivity over all other receptors/
mechanisms tested. In human stomach RQ-00201894 0.1e30
m
M concentration-dependently increased
EFS-evoked contractions (up to 1209%; pEC
50
6.0). At 0.1e10
m
M this activity was usually prolonged. At
higher concentrations (3e30
m
M) RQ-00201894 also caused a short-lasting muscle contraction,
temporally disconnected from the increase in EFS-evoked contractions. RQ-00201894 10
m
M did not
consistently affect submaximal contractions evoked by carbachol. In conclusion, RQ-00201894 potently
and selectively activates the motilin receptor and causes long-lasting facilitation of cholinergic activity in
human stomach, an activity thought to correlate with an ability to increase gastric emptying.
©2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
The gastrointestinal (GI) hormone motilin is released from the
upper gastrointestinal tract during hunger to evoke large,
migrating contractions of the stomach which represent phase III of
a pattern of GI movements during hunger, known as the migrating
motor complex (MMC) (1). Further, this release of motilin may
contribute to the mechanisms by which the sensation of hunger is
initiated (2). Interestingly, however, motilin receptor agonists are
also the target for developing new drugs which cause prolonged
stimulation of gastric emptying of meals. This follows the discovery,
in 1989, that the anti-biotic drug erythromycin is a non-selective
motilin receptor agonist (3), followed by its subsequent ‘off label’
use as a gastric prokinetic agent (e.g. treating patients with gas-
troparesis, helping to control blood glucose levels in diabetic pa-
tients and increasing gastric emptying in patients receiving enteral
feeding or requiring clearance of gastric contents before endoscopy
or emergency surgery and its use in critically ill patients needing
rapid intubation) (1). In addition, azithromycin, another antibiotic
drug which activates the motilin receptor, is used to treat certain
patients with gastro-oesophageal reflux, where increasing gastric
emptying facilitates clearance of acid from the oesophagus (4).
Nevertheless, there is concern that antibiotic drugs should not be
used when there is no infection to treat, since this may contribute
to growing anti-bacterial drug resistance (5). Erythromycin and
azithromycin also have additional actions (e.g. inhibition of puri-
nergic P2X channels and cytochrome 3A4 by erythromycin, and in
rare cases, the potential for cardiac QT prolongation, arrhythmia
and sudden cardiac death by erythromycin and azithromycin) (1, 4,
*Corresponding author.
E-mail address: g.sanger@qmul.ac.uk (G.J. Sanger).
Peer review under responsibility of Japanese Pharmacological Society.
HOSTED BY
Contents lists available at ScienceDirect
Journal of Pharmacological Sciences
journal homepage: www.elsevier.com/locate/jphs
http://dx.doi.org/10.1016/j.jphs.2015.11.004
1347-8613/©2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
Journal of Pharmacological Sciences xxx (2015) 1e6
Please cite this article in press as: Broad J, et al., RQ-00201894: A motilin receptor agonist causing long-lasting facilitation of human gastric
cholinergically-mediated contractions, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.11.004
6, 7). Finally, the optimal doses required to treat patients with
gastroparesis are not rigorously established, with most in-
vestigators now using doses which are lower than those required to
treat infections, an approach which appears to retain gastric pro-
kinetic activity with minimal adverse events such as the induction
of nausea (1).
Progress in identifying a non-antibiotic drug which acts selec-
tively at the motilin receptor has been slow; as yet, no selective
motilin receptor agonist has entered clinical practice. In part, the
slow progress can be attributed to the complex macrolide struc-
tures of erythromycin, azithromycin and related molecules. This
complexity greatly impedes the ability to undertake the precise
structure-activity studies needed to optimise the selectivity of ac-
tion, efficacy and duration of action of a motilin receptor agonist
suitable for clinical use, in addition to the pharmacokinetic char-
acteristics which enable such a molecule to become a successful
drug (8). Thus, the identification of motilin receptor agonists with
simpler structures, including those derived from dihydro-
triazolopyridazine-1,3-dione-based amino acids (9) and more
recently, the low molecular weight receptor agonist, camicinal
(GSK962040 (10)) are now providing improved opportunities to
develop effective motilin receptor agonists and successful drugs.
RQ-00201894 is a new non-macrolide, small molecule motilin
receptor agonist (11). The pharmacology of RQ-00201894 is now
defined in terms of its potency, efficacy and selectivity at the human
motilin receptor. In addition, the ability of RQ-00201894 to facili-
tate gastric cholinergic motor transmission has been characterised
using human isolated stomach. This is an important assay not just
because it reflects the functions of the main excitatory motor
nerves within the stomach, but because it also identifies motilin
receptor agonists which induce either a long-lasting facilitation of
cholinergic activity, more suitable for a gastric prokinetic drug or,
like motilin itself, induce a shorter-lasting facilitation consistent
with its role in mediating phase III MMC activity (12). Some of the
data have previously been presented as meeting abstracts (13, 14).
2. Materials and methods
2.1. Activity of RQ-00201894, motilin and erythromycin at the
human recombinant motilin receptor
CHO cells stably expressing both NFAT/
b
-lactamase reporter
gene and human motilin receptor were seeded into 384-well black/
clear-bottom plates. After overnight incubation, the culture me-
dium was replaced by serum free medium at 37
C for 1.5 h. The
cells were then treated with the motilin receptor agonists at 37
C
for 4.5 h. Afterwards, CCF4/AM, a fluorogenic substrate (Invitrogen)
was added and the cells incubated for 2 h. The fluorescence in-
tensities were measured (Functional Drug Screening System;
Hamamatsu Photonics) with excitation at 405 nm and emission at
465 and 540 nm. The concentration-response curves for RQ-
00201894, motilin, and erythromycin were expressed as a per-
centage of the maximal control activity (50
m
M erythromycin), and
analyzed using GraphPad Prism (GraphPad Software) to obtain EC
50
values. Data represent mean ±S.E.M. of three independent exper-
iments performed in duplicate.
2.2. Evaluation of selectivity of action
The activity of a single concentration of RQ-00201894 (10
m
M)
was examined using a range of 64 different GPCRs, transporters,
enzymes and ion channels expressed in native tissues and cell lines
expressing human or rat recombinant proteins, provided by Cerep
(Cerep, Paris, France), using standard radioligand binding, func-
tional and enzyme assays. Specifically for the human ghrelin
receptor, at which the effects of a range of concentrations of RQ-
00201894 were studied, the receptors were transiently expressed
in HEK293 cells and after seeding into 96 well plates (20,000 cells/
100
m
l/well) their function assessed using a Ca
2þ
influx assay with
fluorescence intensity measured as above, as a ratio of emission
intensities (510 nm from 340 nm excitation/510 from 380 nm). The
concentration-response curves of RQ-00201894 and human ghrelin
were expressed as percentage in proportion with maximal control
activity (1
m
M human ghrelin).
2.3. Human stomach
The method followed that which we have previously described
(12). In brief, segments of stomach were obtained from patients
undergoing surgery for obesity. The study was approved by the East
London Research Ethics Committee 1, (REC reference number 10/
H0703/71, SSA reference number 10/H0703/76), and written
informed consent was obtained from all patients Tissues were
transferred to the laboratory within 2 h after resection in Krebs'
solution (mM: NaCl 121.5, CaCl
2
2.5, KH
2
PO
4
1.2, KCl 4.7, MgSO
4
1.2,
NaHCO
3
25, glucose 5.6) equilibrated with 5% CO
2
and 95% O
2
and
were used immediately or after overnight storage at 4
C in fresh,
oxygenated Krebs' solution.
The mucosa, muscularis mucosa and submucosal plexus were
removed by blunt dissection. Strips of gastric antrum
(3e515 mm) were cut approximately parallel to the circular
muscle fibres and mounted in tissue baths (containing Krebs so-
lution at 35
C, gassed with 5% CO
2
in O
2
) for measurement of
changes in muscle tension using pre-calibrated isometric force
transducers (AD Instruments, Chalgrove, UK) linked to a data
acquisition system (Biopac Inc., CA, USA). After an initial application
of 2 g tension the strips were allowed to recover for 60 min, during
which the bath solutions were changed every 15 min. The strips
were then stimulated via two parallel platinum ring electrodes
connected to a stimulator (STG2008, Scientifica, Uckfield, UK).
Electrical field stimulation (EFS) was applied at 5 Hz for 10 s using
50 V (c.200 mA) and 0.5 m bipolar pulse duration, repeated every
1 min. These parameters evoked monophasic, cholinergically-
mediated contractions, attenuated by simultaneous activation of
nitrergic inhibitory neurons (12), and were applied continuously
until consistent responses were obtained (bath solution changed
every 15 min).
In the experiments to examine the effects of RQ-00201894 on
EFS-evoked contractions, each strip of stomach muscle was
exposed to only a single concentration of RQ-00201894, which was
then left in contact with the tissue for at least 60 min. The following
measurements were taken:
1 Change in baseline muscle tension (expressed as a % of at least
three pre-treatment EFS-induced contractions) and duration of
any change,
2 Maximum change in EFS-evoked contractions (determined by
measuring at least three EFS-induced responses at a given time-
point, expressed as a percentage of the mean of at least three
pre-treatment EFS-induced responses (100%)) and the time
taken to achieve maximum response,
3 The length of time over which the maximum increase in EFS-
evoked contraction was maintained during the continuous
presence of the compound, measuring the time of fade of
response if appropriate,
4 The occurrence of irregular contractions in response to EFS after
application of RQ-00201894, recorded as a simple presence or
absence.
J. Broad et al. / Journal of Pharmacological Sciences xxx (2015) 1e62
Please cite this article in press as: Broad J, et al., RQ-00201894: A motilin receptor agonist causing long-lasting facilitation of human gastric
cholinergically-mediated contractions, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.11.004
In separate experiments to investigate the effects of RQ-
00201894 on contractions evoked by carbachol, a concentration
of 1
m
M carbachol was used (representing the concentration
causing ~50% of maximum contraction; data not shown) and
consistent responses were obtained (5 min contact, repeated at
15 min intervals) before RQ-00201894 was added.
2.4. Statistical analysis
Data obtained from experiments using the recombinant re-
ceptors are expressed as geometric means with 95% confidence
ranges. Curves were fitted using a 4 parameter (log) agonist
response curve. Data obtained from the human stomach experi-
ments are expressed as medians and ranges or as the
mean ±standard error of the mean; n values are the numbers of
patients. Curves were fitted using a 3 parameter (log) agonist
response curve. Changes of baseline tension, EFS magnitude and
responses to carbachol were compared using Wilcoxon signed rank
tests. P<0.05 is considered as statistically significant. All data were
analysed using GraphPad Prism 5.
2.5. Drugs
In the experiments using the recombinant motilin and ghrelin
receptors, RQ-00201894 and erythromycin were dissolved and
diluted with 100% DMSO before final addition to the appropriate
assay buffer Motilin receptor assay: 100 ml of HBSS (1000 ml þ1M
HEPES 20 ml), 5 ml BSA (20 mg/ml) and 1 ml DMSO; Ghrelin re-
ceptor assay: 100 ml of HBSS (1000 ml þ1 M HEPES 20 ml) and
adding to the cells. Human motilin was dissolved with distilled
water before serial dilution using 100% DMSO and assay buffer, as
above. Human ghrelin was dissolved with distilled water. For all
other experiments with recombinant receptors, RQ-00201894 was
Fig. 1. Concentration-response curves of RQ-00201894, human motilin, and
erythromycin at human motilin receptors expressed in CHO cells, obtained using a
b
-lactamase reporter gene assay. Data represent percentage relative to the maximal
control activity at 50
m
M erythromycin and show arithmetic mean ±SEM of three
independent variables.
Fig. 2. Representative experimental records showing the responses of circular muscle strips of human antrum to the continuous presence of 1e30
m
M RQ-00201894. The
contractions are shown in response to EFS (50 V, 0.5 m bipolar pulse duration, 5 Hz) given for 10 s, every 1 min. Note the different response kinetics after application of RQ-
00201894.
J. Broad et al. / Journal of Pharmacological Sciences xxx (2015) 1e63
Please cite this article in press as: Broad J, et al., RQ-00201894: A motilin receptor agonist causing long-lasting facilitation of human gastric
cholinergically-mediated contractions, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.11.004
dissolved in 100% DMSO at 10 mM. In the experiments using hu-
man stomach, all drugs were freshly prepared prior to use. Carba-
chol and RQ-00201894 were dissolved in dH
2
O.
3. Results
3.1. Activity of RQ-00201894 at the recombinant human motilin
receptor
RQ-00201894, motilin and erythromycin were equi-effective as
motilin receptor agonists, the E
max
values for RQ-00201894 and
motilin being similar to the activity evoked by the maximum
concentration of erythromycin (50
m
M) (Fig. 1). In this assay, the
EC
50
values for RQ-00201894, motilin and erythromycin were,
respectively, 0.20 (geometric mean with 95% confidence limits:
0.19e0.22), 0.11 (0.07e0.19) and 69 (51e94) nM; the E
max
values
were 101 (99e103), 97 (95e100)% and 98 (95e101)%; and the Hill
slopes were 1.5 (1.3e1.7); 1.9 (1.5e2.3) and 1.9 (1.6e2.3) all from 3
independent experiments performed in triplicate.
3.2. Selectivity data
RQ-0020189410
m
M had little or no activity when tested against
a Cerep panel of 64 receptors, ion channels and enzymes. In the
binding studies the exception was the Na
þ
channel (IC
50
1.5
m
M). In
the functional assays the exception was the 5-HT
1B
receptor (EC
50
8.6
m
M). Further, RQ-00201894 acted as an agonist at the human
ghrelin receptor but at concentrations approximately 95 times
higher than those required to activate the motilin receptor (EC
50
19
(geometric mean with 95% confidence limits: 11e34) nM at the
ghrelin receptor, compared with 4.9 (2.0e12) nM for ghrelin itself;
n¼3 independent experiments).
3.3. Effect in human stomach
52 strips of human isolated antrum muscle were obtained from
8 patients undergoing sleeve gastrectomies for obesity. The male to
female ratio was 1:1; median age (range) ¼51 (25e60). 3 tissues
were used on the same day as the operation, and 5 were used
following overnight storage in fresh oxygenated Krebs solution at
4
C, following removal of the mucosa.
After achieving consistent responses to EFS (obtained after 208
(189e256) minutes), the application of RQ-00201894 0.1e30
m
M
concentration-dependently increased the amplitude of the EFS-
evoked contractions (Figs. 2 and 3;Table 1), with an E
max
of
1209 ±183% and a pEC
50
¼6.0 ±0.4, n ¼4e6 patients for each
concentration; P¼0.03 at 10
m
M; lower concentrations (0.001,
0.01
m
M) had no consistent activity. At low concentrations
(0.1e10
m
M) this excitatory action of RQ-00201894 was slow in
onset and after reaching maximum, was usually maintained for the
remainder of the experiment; at the highest concentration (30
m
M)
the response faded during the continued presence of the com-
pound (Figs. 2 and 4;Table 1). Notably, the facilitation of the EFS-
evoked responses did not always occur in a regular manner, with
large and small EFS-evoked contractions appearing at irregular
intervals especially at the higher concentrations (Figs. 2 and 4).
Relatively higher concentrations of RQ-00201894 (3e30
m
M)
also consistently evoked a short-lasting muscle contraction (an
E
max
of 292 ±76% and a pEC
50
¼5.9 ±0.6, n ¼4e6 patients for each
concentration; P¼0.03 at 10
m
M; n ¼6) which appeared tempo-
rally disconnected from the increase in EFS-evoked contractions,
reaching maximum more quickly and usually fading completely
before the increase in EFS-evoked contraction had reached
maximum (Figs. 2 and 3;Table 1); the lower concentrations of RQ-
00201894 contracted the muscle in 2 of 5 tissues at 1
m
M, in 3 of 5
tissues at 0.3
m
M and in 2 of 6 tissues at 0.1
m
M.
In separate experiments, RQ-00201894 10
m
M had no consistent
ability to affect the magnitude of contractions evoked by a
submaximally-effective concentration of carbachol (1
m
M; con-
tractions were 83 ±13% of the responses before RQ-00201894
addition; n ¼4, P>0.05).
4. Discussion
RQ-00201894 demonstrated good potency as an agonist at the
human recombinant motilin receptor expressed in CHO cells, as
well as selectivity of action when compared with the higher con-
centrations needed to activate the ghrelin receptor and more
especially, interact with a range of other GPCRs, transporters, ion
channels and enzymes. This selectivity of action as a motilin re-
ceptor agonist contrasts with the non-selective activity for eryth-
romycin, azithromycin and other macrolide structures with
antibiotic and motilin receptor agonist activity (1, 4, 6, 7; see
Introduction for details). The potency of RQ-00201894 as a motilin
receptor agonist in this assay was similar tothat of motilin itself and
>300 times greater than the potency demonstrated by erythro-
mycin. It is likely that RQ-00201894 binds to the orthosteric bind-
ing site of the motilin receptor, as increasing concentrations of GM-
109 (15) have been previously demonstrated to cause surmount-
able rightward shifts in the concentration-response curves to RQ-
00201894 in the NFAT/
b
-lactamase assay (13).
In human isolated gastric antrum, RQ-00201894 0.1e30
m
M
concentration-dependently facilitated cholinergically-mediated
contractions. This activity was slow to reach maximum and once
achieved, was usually prolonged (with the exception of 30
m
M, the
highest concentration tested, where fade was clearly observed
during the experiments). Notably, the potency of RQ-00201894 in
Fig. 3. Effects of RQ-00201894 on contractions to EFS in circular muscle strips from
human gastric antrum. Panel A displays the concentration relationship of the facili-
tation of EFS by RQ-00201894 0.001e30
m
M. Panel B displays the concentration rela-
tionship of the increase in baseline muscle tension induced by RQ-00201894. EFS
(50 V, 0.5 m bipolar pulse duration, 5 Hz) was given for 10 s, every 1 min. n ¼4e6
patients for each concentration; drugs were applied non-cumulatively.
J. Broad et al. / Journal of Pharmacological Sciences xxx (2015) 1e64
Please cite this article in press as: Broad J, et al., RQ-00201894: A motilin receptor agonist causing long-lasting facilitation of human gastric
cholinergically-mediated contractions, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.11.004
the human gastric antrum was lower than that when tested at the
human recombinant receptor (respectively, pEC
50
values of 6.0 and
9.5). Similar differences in potencies have previously been reported
for motilin, erythromycin, azithromycin and camicinal when
comparing data obtained using recombinant receptors and human
isolated stomach (4, 12), perhaps reflecting the additional need for
these compounds to penetrate into the muscle and reach the
motilin receptors on the cholinergic nerves. RQ-00201894 also
evoked small increases in muscle tension. These contractions
occurred consistently at concentrations (3e30
m
M) which were
higher than the threshold concentrations which increased EFS-
evoked contractions, although at lower concentrations somedbut
not alldtissues contracted in response to RQ-00201894. Notably,
the contractions were relatively rapid in onset and usually faded
completely before RQ-00201894 had maximally increased EFS-
evoked contractions. Such activity is consistent with the actions
of other motilin receptor agonists in human stomach (4, 12). The
different neuronal and muscle responses are thought to represent
the main motor pathways by which low doses of motilin receptor
agonists increase gastric emptying (by facilitation of cholinergic
activity) or when given at higher doses, directly contract the
stomach muscle to induce early satiety and nausea (exemplified by
the use of erythromycin at the relatively high doses required for
antibiotic treatment) (1).
In human stomach, RQ-00201894 is likely to increase cholin-
ergic activity by facilitating the release of acetylcholine from the
intrinsic cholinergic neurons of the stomach. Thus, in common with
other motilin receptor agonists (4, 12) a high concentration of RQ-
00201894 had no consistent ability to modulate the contractions
evoked by a submaximally-effective concentration of carbachol.
Notably, the increase in cholinergic activity by RQ-00201894 can
occur in a prolonged manner. A broadly-similar long-lasting ac-
tivity has previously been reported for erythromycin and azi-
thromycin, drugs used to treat gastroparesis or increase gastric
emptying, and also camicinal, under development for treatment of
these disorders (4, 12; see Introduction for more detail). By contrast,
a relatively short-lasting activity of motilin is consistent with its
physiological role in inducing the migrating phase III contractions
of the MMC (1, 4, 12, 16, 17). The explanation for these different
profiles of activity for motilin and the synthetic motilin receptor
agonists is unclear but ‘functional’or ‘biased’agonism has been
hypothesised, with the molecules acting at one or more binding
Table 1
Summary of effects of RQ-00201894 on baseline muscle tension and on neuronally-
mediated contractions during electrical field stimulation in human isolated colon.
Muscle [n] Nerve [n]
Effective concentrations 0.3e30
m
M[5e6] 0.1e30
m
M[5e6]
pEC
50
(mean ±standard error)
5.9 ±0.5 [4e6] 6.0 ±0.4 [4e6]
E
max
(%)
(mean ±standard error)
292 ±76* [4e6] 1209 ±183 [4e6]
T
max
at 10
m
M (min)
(median, with range)
10 (5e16) [6] 44 (10e51) [6]
T
fade
at 10
m
M (min)**
(median, with range)
29 (12e51) [6] not observed [5] except
in one tissue where the
T
max
was short (10 min)
and fade occurred
within 17 min
The values shown are determined from a concentration-response curve for RQ-
00201894 (0.001e30
m
M) constructed by adding single concentrations of the
compound to separate tissues; n ¼4e6 patients for each concentration. * % of
contraction amplitude evoked by EFS prior to addition of RQ-00201894. ** the time
at which the response faded is recorded as the point at which the increase in muscle
tension had completely returned to baseline, or for EFS as the point at which the
facilitation had declined by 50%.
Fig. 4. Duration of facilitation by RQ-00201894 of EFS-evoked contractions in circular muscle strips from human gastric antrum. For each tissue studied, the mean of three
consecutive contractions were calculated consecutively throughout the experiment. Panels AeD shows the time course of the mean ±S.E.M. response to application of RQ-
00201894 1e30
m
M respectively. Note the different response kinetics and the irregularity of contractions during fade of response to RQ-0 0201894 (in which small contractions
occurred in-between larger contractions), particularly at the higher concentrations. EFS (50 V, 0.5 m bipolar pulse duration, 5 Hz) was given for 10 s, every 1 min. n ¼5e6 patients
for each concentration as displayed on panels.
J. Broad et al. / Journal of Pharmacological Sciences xxx (2015) 1e65
Please cite this article in press as: Broad J, et al., RQ-00201894: A motilin receptor agonist causing long-lasting facilitation of human gastric
cholinergically-mediated contractions, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.11.004
sites on the motilin receptor to differentially influence different
intracellular signalling pathways (18). Interestingly, the increase in
EFS-evoked contractions caused by RQ-00201894 was not always
consistent, especially at the higher concentrations tested, and the
contraction amplitudes became irregular. The reason why irregular
contractions should occur during EFS is unknown. One possibility,
discussed by Broad et al (12), is that the activities of different groups
of interstitial cells of Cajal (ICCs) within the intestinal wall become
uncoordinated so that overall, the baseline muscle electrical ac-
tivity rises and falls at a frequency which is markedly slower than
the frequency of EFS, influencing the amplitudes of the contractions
evoked by EFS. An ability of different groups of ICCs to move out of
synchrony with each other has previously been observed in human
isolated jejunum, a phenomenon linked to disordered GI move-
ments (19).
In other studies with RQ-00201894 in conscious fasted dogs, the
compound has been shown to induce high-amplitude contractions
of gastric antrum and duodenum (similar to the phase III-like ac-
tivity of the migrating motor complex (MMC) during hunger,
mediated by endogenous motilin) without causing vomiting and
even after suppression of spontaneous contractility by clonidine.
Further, RQ-00201894 increased gastric emptying of meals con-
taining acetaminophen in conscious dogs and cynomolgus mon-
keys (13, 20). Notably, the dog and human motilin receptors are
phylogenetically distinct (21) with only 71% protein sequence
identity between the two receptors and up to 2 log lower potencies
for different motilin receptor agonists at the dog receptor (22, 23).
Further, motilin receptor immunoreactivity has been detected
within the enteric nervous system (ENS) but not in the muscle of
dog stomach (21) whereas in humans motilin receptor immuno-
reactivity has been detected in both, with low concentrations of
motilin facilitating cholinergic activity and higher concentrations
directly contracting the muscle (12); these differences suggest
significantly different dynamics of the response to motilin between
the two species.
In summary, RQ-00201894 demonstrates good agonist potency
and selectivity at the human motilin receptor and in human
stomach, facilitates cholinergic activity in a manner that is broadly
consistent with other non-peptide motilin receptor agonists. These
data suggest that RQ-00201894 will promote human gastric
emptying and should be evaluated as a potential new drug for
treatment of disorders such as gastroparesis and in other groups of
patients where there is a need to increase gastric emptying.
Conflicts of interest
NT, MT, MS and TY are employees of RaQualia. GJS received
funding from RaQualia for JB to conduct the human stomach
studies. AG, UP and KM report no conflict of interest.
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J. Broad et al. / Journal of Pharmacological Sciences xxx (2015) 1e66
Please cite this article in press as: Broad J, et al., RQ-00201894: A motilin receptor agonist causing long-lasting facilitation of human gastric
cholinergically-mediated contractions, Journal of Pharmacological Sciences (2015), http://dx.doi.org/10.1016/j.jphs.2015.11.004
