Acotiamide hydrochloride (Z-338), a new selective acetylcholinesterase inhibitor, enhances gastric motility without prolonging QT interval in dogs: comparison with cisapride, itopride, and mosapride.

Acotiamide hydrochloride (acotiamide; N-[2-[bis(1-methylethyl) amino]ethyl]-2-[(2-hydroxy-4,5-dimethoxybenzoyl) amino] thiazole-4-carboxamide monohydrochloride trihydrate, Z-338) has been reported to improve meal-related symptoms of functional dyspepsia in clinical studies. Here, we examined the gastroprokinetic effects of acotiamide and its antiacetylcholinesterase activity as a possible mechanism of action in conscious dogs. Acotiamide increased postprandial gastric motor activity in conscious dogs with chronically implanted force transducers and, like itopride, mosapride, and cisapride, exhibited gastroprokinetic activity in these dogs. Furthermore, acotiamide improved clonidine-induced hypomotility and delayed gastric emptying. Acotiamide-enhanced postprandial gastroduodenal motility was suppressed completely by pretreatment with atropine, a muscarinic receptor antagonist. In in vitro studies, acotiamide enhanced acetylcholine- but not carbachol-induced contractile responses of guinea pig gastric antrum strips. Moreover, like itopride and neostigmine, acotiamide inhibited recombinant human and canine stomach-derived acetylcholinesterase (AChE) activity in vitro. The mode of the AChE inhibitory action of acotiamide was selective and reversible. Unlike itopride or mosapride, acotiamide showed no affinity for dopamine D(2) or serotonin 5-HT(4) receptors. With regard to cardiovascular side effects, unlike cisapride, acotiamide did not affect myocardial monophasic action potential duration, QT interval, or corrected QT interval in anesthetized dogs. These results suggest that acotiamide stimulates gastric motility in vivo by inhibiting AChE activity without affecting QT interval. Acotiamide thus represents a beneficial new drug for the treatment of functional dyspepsia involving gastric motility dysfunction, with differences from other prokinetic agents.

House musk shrew (Suncus murinus, order: Insectivora) as a new model animal for motilin study.

Although many studies have demonstrated the action of motilin on migrating motor complex by using human subjects and relatively large animals, the precise physiological mechanisms of motilin remain obscure. One reason for the lack of progress in this research field is that large animals are generally not suitable for molecular-level study. To overcome this problem, in this study, we focused on the house musk shrew (Suncus murinus, order: Insectivora, suncus named as laboratory strain) as a small model animal, and we present here the results of motilin gene cloning and its availability for motilin study. The motilin gene has a high homology sequence with that of other mammals, including humans. Suncus motilin is predicted to exist as a 117-residue prepropeptide that undergoes proteolytic cleavage to form a 22-amino-acid mature peptide. The results of RT-PCR showed that motilin mRNA is highly expressed in the upper small intestine, and low levels of expression were found in many tissues. Morphological analysis revealed that suncus motilin-producing cells were present in the upper small intestinal mucosal layer but not in the myenteric plexus. Administration of suncus motilin to prepared muscle strips of rabbit duodenum showed almost the same contractile effect as that of human motilin. Moreover, suncus stomach preparations clearly responded to suncus or human motilin stimulation. To our knowledge, this is the first report that physiological active motilin was determined in small laboratory animals, and the results of this study suggest that suncus is a suitable model animal for studying the motilin-ghrelin family.

Characterization of multinuclear hepatocytes induced in rats by mitemcinal (GM-611), an erythromycin derivative.

Mitemcinal is an erythromycin derivative with motilin agonistic action, developed as a gastrointestinal motor-activating agent. The characteristics of mitemcinal-induced multinuclear hepatocytes (MNHs, hepatocytes with three or more nuclei per cell) from detailed morphological observations together with the results of a study on the mechanisms of MNH formation by combining cytocentrifuge preparations with 5-bromo-2'-deoxyuridine cumulative labeling are reported. MNHs were observed only in rats in the high-dose groups of the subchronic study, with a higher incidence in females and reversibility after twenty-eight days of drug withdrawal, but not observed in dogs. In the chronic study, the incidence increased relative to the dose. Histopathologically, MNHs were preferentially observed in the centrilobular zone, without nuclear atypia or mitotic figures. In the cell kinetic study, the labeling pattern of MNHs included all-positive, all-negative, and mixed labeling patterns of nuclei. The all-negative pattern indicated that the cells were formed by fusion of nondividing cells. The current results indicate that the cell kinetic approach effectively demonstrated the mechanism of mitemcinal-induced MNHs as fusion of hepatocytes and that drug-induced disturbance of mitosis is not involved in the multinucleation of MNHs by mitemcinal.

Effects of mitemcinal (GM-611), an orally active erythromycin-derived prokinetic agent, on delayed gastric emptying and postprandial glucose in a new minipig model of diabetes.

AIMS: This study was conducted to evaluate the suitability of a new minipig model for investigating aspects of diabetes such as delayed gastric emptying and glucose metabolism abnormalities, and to test the effects of mitemcinal (GM-611), an orally active erythromycin-derived motilin receptor agonist, on gastric emptying and postprandial glucose in normal and diabetic minipigs. METHODS AND RESULTS: Intravenous injection of 300 mg/kg streptozotocin (STZ) to 5-week-old minipigs induced moderate hyperglycemia (about 200 mg/dl) for >80 weeks without insulin treatment. Decreased insulin production (P<.05), increased area under the glucose curve (P<.05), and slower glucose disappearance (P<.05) were demonstrated, and there was no severe inhibition of body weight gain, liver failure, or renal failure. Gastric emptying was significantly delayed in diabetic minipigs (P<.05) at 80 weeks, but not at 40 weeks, post-STZ. Oral administration of mitemcinal (5 mg/kg) at 80 weeks accelerated gastric emptying and induced a similar postprandial glucose profile in normal and diabetic minipigs with delayed gastric emptying. CONCLUSIONS: The new diabetic minipig model showed suitability for investigating diabetes, gastric emptying, and plasma glucose excursions. Since delayed gastric emptying and irregular plasma glucose excursions are characteristic of diabetic gastroparesis, the accelerating and regulating effects of mitemcinal on this model add to the existing evidence that mitemcinal is likely to be useful for treating diabetic gastroparesis.

Assessment of the carcinogenic potential of mitemcinal (GM-611): increased incidence of malignant lymphoma in a rat carcinogenicity study.

Mitemcinal is an erythromycin derivative, which acts as an agonist of the motilin receptor. For assessment of the carcinogenicity of mitemcinal, we conducted a short-term carcinogenicity study in p53 (+/-) C57BL/6 mice and a 104-week carcinogenicity study in CD(SD)IGS rats. There was no evidence of a carcinogenic potential in mouse when administered for 26 consecutive weeks at levels up to 250 mg/kg/day. In the rat study, an increased incidence of lymphoma was noted in 5/60 males and 8/60 females of the high dose group (60 mg/kg/day) compared to 1/60 and 0/60 in control males and females, respectively, with statistical significance in females. Rat lymphomas include different immunomorphologic types (T- or B-cell lineage). Immunohistochemical analysis revealed that lymphomas from mitemcinal-treated rats and spontaneous cases were of T-cell lineage. The overall weight of evidence suggests that the incidence of spontaneous lymphoma was enhanced in the rat study. They also indicate that the increased incidence of lymphomas was based on a non-genotoxic effect with a threshold dose-response and that the tumorigenesis was based on the strain or species specificity of background factors. The high dose in the rat study is approximately 1600-fold higher (AUC) than that of the clinical dose, a sufficient margin of safety for the clinical dose. We conclude that the risk of carcinogenesis due to mitemcinal in humans can be considered to be minimal and is to represent an acceptable risk for the continued administration of mitemcinal to humans.

Mitemcinal (GM-611), an orally active motilin receptor agonist, improves delayed gastric emptying in a canine model of diabetic gastroparesis.

1. The aim of the present study was to evaluate the effects of mitemcinal (GM-611), an orally active motilin receptor agonist, on delayed gastric emptying in a canine model of diabetic gastroparesis and to compare these effects with those of cisapride. 2. Moderate hyperglycaemia was induced by a single intravenous injection of a mixture of streptozotocin (30 mg/kg) and alloxan (50 mg/kg). Dogs that maintained moderate hyperglycaemia (fasting plasma glucose 200-300 mg/dL) without insulin treatment were selected and gastric emptying in these dogs was determined by the paracetamol method. 3. One year after the onset of diabetes, there was no difference in the gastric emptying of normal and diabetic dogs. However, after 5 years, the diabetic dogs showed delayed gastric emptying. The motor nerve conduction velocity of the tibial nerve was significantly lower in diabetic dogs compared with normal dogs at both time points. 4. Histopathological examination at the end of the study showed that there were fewer nerve fibres in both dorsal vagal and tibial nerves of diabetic dogs compared with normal dogs. The onset of delayed gastric emptying is thought to have occurred gradually, in parallel with abnormal autonomic nerve function induced by the long period of moderate hyperglycaemia. 5. Oral administration of mitemcinal (0.125, 0.25 or 0.5 mg/kg) dose-dependently accelerated delayed gastric emptying, significant at 0.5 mg/kg, in diabetic dogs, whereas cisapride (1, 3 or 10 mg/kg) had no significant effect. These results add to the existing evidence that mitemcinal is likely to be useful for treating diabetic gastroparesis.

Effects of oral mitemcinal (GM-611), erythromycin, EM-574 and cisapride on gastric emptying in conscious rhesus monkeys.

We assessed and compared the effects of oral mitemcinal (an orally active, erythromycin-derived motilin-receptor agonist; Code name: GM-611), erythromycin, EM-574 and cisapride on gastric emptying in conscious Rhesus monkeys using the acetaminophen method. Mitemcinal and erythromycin induced significant, dose-dependent increases in indices of gastric emptying, but mitemcinal required a much lower dose for the same effect. Cisapride induced a bell-shaped dose response, and EM-574, a potent erythromycin derivative and originally developed as an enteric coated formulation, had little effect when it was given orally uncoated. Since our previous study showed that response to motilin is similar in Rhesus monkeys and humans, these results suggest that oral mitemcinal may be effective for the treatment of symptoms in human disorders related to delayed gastric emptying (e.g., functional dyspepsia and gastroparesis). Combined with the results of other studies, these results suggest that mitemcinal may be able to replace the withdrawn drug, cisapride, as the drug of choice for treating delayed gastric emptying.

Oral mitemcinal (GM-611), an erythromycin-derived prokinetic, accelerates normal and experimentally delayed gastric emptying in conscious dogs.

1. We examined effects of orally administered mitemcinal, an erythromycin-derived motilin agonist, on gastric emptying and antroduodenal motility in conscious normal dogs and conscious dogs with experimentally delayed gastric emptying. For comparison, we also examined the effects of orally administered cisapride. 2. Gastric emptying was assessed by adding paracetamol to the test meal and determining three of its pharmacokinetic parameters as indices of gastric emptying. Antroduodenal motility was assessed from the output of force transducers chronically implanted in the gastric antrum and duodenum. 3. In normal dogs, mitemcinal (0.25, 0.5 and 1 mg/kg) dose-dependently accelerated gastric emptying, significantly increasing all three indices at doses of 0.5 and 1 mg/kg; cisapride (1, 3 and 10 mg/kg) had no significant effect. Mitemcinal also dose-dependently stimulated antroduodenal motility in the interdigestive and digestive states. Cisapride, at 100-fold the dose, produced similar effects in the interdigestive state, but mixed results in the digestive state. 4. In dogs with delayed gastric emptying induced by subcutaneous clonidine (0.03 mg/kg), mitemcinal (0.25, 0.5 and 1 mg/kg) dose-dependently improved delayed gastric emptying, significantly increasing two of three indices at a dose of 1 mg/kg. Cisapride (1, 3 and 10 mg/kg) caused non-significant increases in the indices of gastric emptying, with roughly bell-shaped dose-response curves. The highest dose of mitemcinal (1 mg/kg) also stimulated antroduodenal motility. 5. In dogs with delayed gastric emptying induced by vagotomy, mitemcinal (0.125, 0.25 and 0.5 mg/kg) dose-dependently improved delayed gastric emptying, significantly increasing all three indices at doses of 0.25 and 0.5 mg/kg. Cisapride (3 mg/kg) restored the indices to roughly prevagotomy levels, but none of the increases was significant. Mitemcinal, at a dose of 0.25 mg/kg, also stimulated antroduodenal motility. 6. Because delayed gastric emptying is the basic characteristic of gastroparesis, the fact that mitemcinal accelerated gastric emptying in dogs with normal and delayed gastric emptying much more robustly than cisapride adds to the evidence that mitemcinal is likely to be useful for the treatment of patients with gastroparesis.

Nonlinear intestinal pharmacokinetics of mitemcinal, the first acid-resistant non-peptide motilin receptor agonist, in rats.

The objective was to investigate the mechanism of nonlinear pharmacokinetics of mitemcinal, the first acid-resistant non-peptide motilin agonist, in rats. Super-proportional increases of the cumulative rates of radioactivity excreted into bile and urine following oral administration of [3H]-mitemcinal suggested nonlinear absorption of mitemcinal in rats. To evaluate the fraction dose absorbed (Fa) and intestinal availability (Fg), [3H]-mitemcinal was orally administrated to rats, and tritium radioactivity and unchanged mitemcinal concentration were determined in the portal blood. Fa values for 0.2 mg/kg1, 0.5 mg/kg, and 5.0 mg/kg dose groups were 0.314, 0.353, and 0.569, respectively. Corresponding Fg values were 0.243, 0.296, and 0.513, respectively. In Caco-2 experiments, the permeation of [3H]-mitemcinal in the secretory direction was larger than in the absorptive direction and was inhibited by P-glycoprotein (P-gp) substrate digoxin. The results indicate that the saturation of P-gp-mediated membrane permeation and intestinal metabolism causes the nonlinear pharmacokinetics of mitemcinal in rats.

Preclinical electrophysiology assays of mitemcinal (GM-611), a novel prokinetic agent derived from erythromycin.

Mitemcinal (GM-611) is a novel erythromycin-derived prokinetic agent that acts as an agonist at the motilin receptor. Erythromycin has shown QT prolongation and torsades de pointes (TdP) in humans and cisapride, a second class of prokinetic agents typified by the 5-HT(4) receptor agonist, has been terminated due to TdP. In this study an extended series of safety pharmacology protocols and evaluations have been undertaken to assess the potential risk of mitemcinal on QT prolongation or proarrhythmic effects. Mitemcinal and its metabolites, GM-577 and GM-625, inhibited the human ether-a-go-go-related gene (HERG) tail current in a concentration-dependent manner with IC(50) values of 20.2, 41.7, and 55.0 microM, respectively. Administration of 10 mg/kg mitemcinal in anesthetized guinea pigs resulted in a slight prolongation of the monophasic action potential (MAP) duration during atrial pacing at the plasma concentration of mitemcinal 1.1 microM, with low maximum increases in MAPD(70) (6.6%) and MAPD(90) (4.6%) relative to vehicle. A 10-min infusion of 20 mg/kg of mitemcinal in a proarrhythmic rabbit model did not evoke TdP even when QT and corrected QT (QTc) intervals were significantly prolonged. In this study, the Cmax plasma-free concentration of mitemcinal indicates that the prolongation was more than 400-fold that of the therapeutic dose. Our findings of a wide safety margin and the absence of TdP within this margin suggest that mitemcinal may provide sufficient safety in clinical use.

Hemodynamic and electrophysiological effects of mitemcinal (GM-611), a novel prokinetic agent derived from erythromycin in a halothane-anesthetized canine model.

Mitemcinal (GM-611) is a novel erythromycin-derived prokinetic agent that acts as an agonist at the motilin receptor. We investigated the QT-prolonging effects of mitemcinal using a halothane-anesthetized canine model. Intravenous administration of mitemcinal at doses of more than 8.3 mg/kg per 10 min significantly prolonged the QT interval corrected by Fridericia's corrections. Mitemcinal exhibited a bradycardiac effect and produced significantly greater prolongation in monophasic action potential duration (MAP(90)) at sinus rhythm compared with MAP(90) at pacing and showed reverse use-dependent prolongation of repolarization, suggesting that the negative chronotropic effect of mitemcinal potentiates the prolongation of the repolarization period. A technique using MAP/pacing electrodes allowed measurements of both MAP(90) and effective refractory period (ERP) simultaneously at the same ventricular site. Although mitemcinal slightly prolonged the MAP(90(CL400)) and ERP in comparison with the control group at the dose of 25 mg/kg per 10 min, the terminal repolarization period, the difference between MAP(90(CL400)) and ERP, did not increase suggesting the absence of a proarrhythmic effect even with a 7000-fold for the therapeutic blood concentration as free level. The electrophysiological results from mitemcinal in this study indicate that the risk of serious arrhythmia such as torsades de pointes, a major clinical concern related to QT interval prolongation, might be low.

Effects of mitemcinal (GM-611), an acid-resistant nonpeptide motilin receptor agonist, on the gastrointestinal contractile activity in conscious dogs.

The effects of mitemcinal (GM-611) on the gastrointestinal contractile activity were investigated using chronically implanted force transducers in conscious dogs and were compared with the effects of porcine motilin (pMTL), EM-523 and EM-574. In the interdigestive state, intravenous and oral administration of mitemcinal, EM-523 and EM-574 induced the gastrointestinal contractile activity in a manner similar to pMTL. The contractile activity caused by mitemcinal was suppressed by continuous intravenous infusion of a motilin receptor antagonist. In the digestive state, intravenous and oral administration of mitemcinal, EM-523 and EM-574 also stimulated the gastrointestinal contractile activity. Mitemcinal, EM-523 and EM-574 given intravenously increased the gastric contractile activity in a similar dose range; however, mitemcinal was approximately 10 times more potent than EM-523 and EM-574 when administered orally in the digestive state. These results indicate that the mitemcinal-induced gastrointestinal contractile activity operates via motilin receptors and possesses a higher activity than EM-523 and EM-574 when administered orally in conscious dogs in the digestive state. Mitemcinal may therefore be useful in the treatment of several gastrointestinal disorders involving dysmotility, such as gastroparesis and functional dyspepsia, even when administered orally.

Effects of motilin and mitemcinal (GM-611) on gastrointestinal contractile activity in rhesus monkeys in vivo and in vitro.

Neither the presence of motilin receptors nor their action has been investigated in monkeys. The object of this study was to determine the effects of motilin and mitemcinal (GM-611), an erythromycin derivative, on the gastrointestinal tract in rhesus monkeys in vivo and in vitro. In in vivo investigations in conscious monkeys, both motilin and mitemcinal induced migrating motor complex-like contractions in the interdigestive state and also accelerated gastric emptying. In in vitro investigations, the presence of motilin receptors in the gastrointestinal tract was demonstrated by receptor binding assays. Motilin and mitemcinal contracted isolated duodenum strips in a concentration-dependent manner. In conclusion, rhesus monkeys may be useful for studying the physiological and pharmacological roles of the motilin agonistic mechanism because they show reactivity to motilin both in vivo and in vitro.

In vitro pharmacological characterization of mitemcinal (GM-611), the first acid-resistant non-peptide motilin receptor agonist, in smooth muscle of rabbit small intestine.

The pharmacological properties of mitemcinal (GM-611), the first acid-resistant non-peptide motilin agonist, were investigated in the smooth muscle of the rabbit small intestine and compared with porcine motilin (pMTL), erythromycin A (EMA) and its derivatives (EM-523, EM-574 and ABT-229). Mitemcinal, pMTL, EMA, EM-523, EM-574 and ABT-229 produced concentration-dependent contractions with approximately the same maximum contractions in the isolated rabbit duodenum longitudinal strips. The contractile response to mitemcinal or pMTL was competitively inhibited by a selective motilin antagonist, GM-109. The pA(2) values for GM-109 as an antagonist of mitemcinal and pMTL showed approximately the same values. However, the concentration-dependent contractile responses to mitemcinal or pMTL were not affected by pretreatment with atropine, tetrodotoxin, hexamethonium, naloxone or tropisetron. The removal of calcium ions from the medium and pretreatment with verapamil greatly suppressed the contractions induced by mitemcinal and pMTL. The contractile response to mitemcinal was not affected by preincubation in acidic solutions, while those of EM-523, EM-574 and ABT-229 were strongly diminished in the same condition. Mitemcinal as well as other motilin agonists displaced (125)I-pMTL bound to a homogenate of the rabbit duodenum muscle tissue. The displacement curves of all these compounds were parallel. These results indicate that mitemcinal is a selective and full motilin receptor agonist in the smooth muscle of the rabbit small intestine and that this agent has an excellent acid-resistant property.

Effects of nitric oxide synthase inhibitor on the digestive system measured by simultaneous monitoring of gastric motility, gastric emptying activity and postprandial pancreaticobiliary secretion in dogs.

Relationships between the NO synthase inhibitor and gastric and pancreaticobiliary functions measured simultaneously in the digestive state have been little studied. The aim of this study was to estimate the effect of NO synthase inhibitor on integrated digestive function in conscious dogs. A strain gauge force transducer was implanted on the gastric antrum of 6 mongrel dogs to measure gastric contractile activity and two duodenal cannulas were inserted into the proximal and distal sites to measure the gastric emptying rate and the pancreaticobiliary output into the duodenum using our novel method. Postprandial pancreatic and biliary secretion were presented as amylase and bile acid activity, respectively. Furthermore, a cervical cannula was placed into the superior vena cava as a route for the administration of NO synthase inhibitor, N omega-nitro-L-arginine (L-NNA), at a dose of 2.5 mg/kg-h. In a group given L-NNA, gastric contractile activity after ingestion was significantly enhanced, but the emptying rates of gastric solids and liquids were significantly suppressed in comparison with the control. The mean 0-1 h amylase integrated output was significantly (P < 0.05) decreased in comparison with the control, and the mean bile acid integration of 0-1 h output was also significantly (P < 0.01) decreased. A possible explanation for this observation is that smaller volumes of nutrient are delivered into the duodenum; however, it could also be that postprandial pancreaticobiliary secretion is inhibited by an alteration of blood flow or by a change in contractions of the sphincter of Oddi after the administration of L-NNA.

Relationship between intraduodenal 5-hydroxytryptamine release and interdigestive contractions in dogs.

BACKGROUND: 5-hydroxytryptamine (5-HT) is released into the intestinal lumen during the fasting state. However, the relationship between the intraduodenal 5-HT and the interdigestive cyclic motor activity in conscious dogs is unclear. AIM: To correlate intraduodenal 5-HT concentrations with the interdigestive gastroduodenal migrating motor complex (MMC). METHODS: 6 dogs were implanted with 2 force transducers for recording gastroduodenal contractions and 2 catheters for measuring duodenal volume by a non-absorbable marker perfusion technique. Intraduodenal 5-HT concentrations were determined by high performance liquid chromatography at 5-min intervals. RESULTS: During fasting, gastroduodenal motor activity cycled as the MMC; luminal 5-HT concentrations and total outputs varied cyclically in temporal association with the MMC. Mean 5-HT concentrations peaked during phase II (P<0.05 vs. phase I and III), and 5-HT outputs during phases II or III were greater than during phase I (P<0.05). Exogenous motilin (0.3 microg/kg-hr, IV) stimulated 5-HT release into the duodenal lumen with peak values (P<0.05) during motilin-induced phase II and III. Gastroduodenal motor activity was not altered, however, during exogenous intraduodenal administration of 5-HT (300 ng/mL-min). CONCLUSIONS: 5-HT is released cyclically into the duodenal lumen in close temporal association with the MMC, but its physiologic significance in regulation of gastroduodenal motility is unknown.

Effect of motilin on endogenous release of insulin in conscious dogs in the fed state.

The aim was to investigate the insulin-releasing activity of motilin during and after feeding. A single intravenous bolus injection of motilin (0.01-0.3 microg/kg) dose-dependently stimulated endogenous release of insulin in the postprandial state. The insulin-releasing activity of motilin in the fed state was completely abolished by pretreatment with atropine or hexamethonium and was partly inhibited by ondansetron. Truncal vagotomy also greatly suppressed the motilin-induced insulin release. While phentolamine significantly enhanced insulin release in response to motilin, propranolol significantly inhibited this response in both states. The motilin-induced insulin release in the fed states was not accompanied by any changes in glucose concentrations. In conclusion, while the physiological significance remains unclear, these results indicate that physiological doses of motilin stimulate endogenous release of insulin via a vagally cholinergic muscarinic pathway, and that adrenergic and 5-hydroxytryptamine3 receptors are also involved in this response, in the dog.