μ-opiate receptor; kappa-opiate receptor; delta-opiate receptor
Mu opioid receptor, Ki=120 nM (rat cerebral cortex membrane preparation) [1]
Kappa opioid receptor, Ki=85 nM (rat cerebral cortex membrane preparation) [1]
Delta opioid receptor, Ki=320 nM (rat cerebral cortex membrane preparation) [1]
Metabolite N-monodesmethyltrimebutine: Ki=65 nM (Mu receptor), Ki=42 nM (Kappa receptor), Ki=180 nM (Delta receptor) [2]

In the rat cerebral cortex membrane receptor binding assay, Trimebutine had the highest affinity for Kappa receptor (Ki=85 nM), followed by Mu receptor (Ki=120 nM), and low affinity for Delta receptor (Ki=320 nM) [1]
In the guinea pig ileum longitudinal muscle preparation assay, Trimebutine concentration-dependently inhibited electrically induced contraction with an EC50=0.8 μM, and this effect was partially blocked by naloxone (blockade rate about 60%) [1]
In the mouse vas deferens preparation assay, 1-10 μM Trimebutine inhibited electrically induced contraction with an EC50=3.2 μM, and this effect was completely reversed by naloxone [1]
In in vitro culture of human colonic smooth muscle cells, 10 μM Trimebutine regulated cellular calcium signaling and inhibited KCl-induced calcium influx, with the calcium concentration decreased by 45% compared to the model group [2]
The in vitro receptor binding activity of the metabolite N-monodesmethyltrimebutine was stronger than that of the parent drug, with higher selectivity for Mu and Kappa receptors [2]

In the rat gastrointestinal motility assay, oral administration of 20 mg/kg Trimebutine significantly accelerated gastric emptying rate by 38% compared to the control group, and shortened small intestinal transit time by 25% [2]
In the mouse acetic acid writhing analgesia assay, the ED50 of intraperitoneally injected Trimebutine was 45 mg/kg, the analgesic rate reached 52% at 60 mg/kg, and the effect lasted for about 2 hours [1]
In the rat colonic spasm model, oral administration of 30 mg/kg Trimebutine relieved colonic smooth muscle spasm, with the spasm index decreased by 63% compared to the model group, and no obvious inhibition on normal colonic motility [2]
In clinical studies, Trimebutine was administered orally at 300 mg daily (divided into 3 doses) to patients with functional dyspepsia. After 4 weeks, the relief rate of bloating and abdominal pain was 72%, and the improvement rate of delayed gastric emptying was 65% [3]
In this clinical application, it also improved the alternating symptoms of diarrhea and constipation in patients with irritable bowel syndrome, with an overall effective rate of 68% [3]

Rat cerebral cortex membrane receptor binding assay: Rat cerebral cortex membrane preparations were prepared and co-incubated with radiolabeled Mu, Kappa, and Delta receptor-specific ligands respectively. Gradient concentrations of Trimebutine were added to compete for binding sites. After incubation at 37°C for 60 minutes, bound and free ligands were separated. Binding affinity was quantified by radioactivity counting, and Ki values for each receptor were calculated [1]
Guinea pig ileum longitudinal muscle contraction inhibition assay: Guinea pig ileum longitudinal muscle strips were isolated and equilibrated in nutrient solution for 40 minutes. After stable contraction was induced by electrical stimulation, gradient concentrations of Trimebutine were added, and changes in contraction amplitude were recorded to calculate EC50. Naloxone was then added to observe the reversal degree of the contraction inhibition effect [1]

Human colonic smooth muscle cell calcium signaling detection: Human colonic smooth muscle cells were seeded in culture plates loaded with fluorescent probes. After culturing to confluence, they were pre-incubated with 5-20 μM Trimebutine for 30 minutes, then KCl was added to stimulate calcium influx. Changes in intracellular calcium concentration were detected by a fluorescence imaging system to quantify the inhibitory effect of the drug on calcium influx [2]

Rat gastrointestinal motility assay: Male SD rats were fasted for 12 hours and randomly grouped. The experimental group was orally administered 20 mg/kg Trimebutine, with the drug dissolved in normal saline containing 5% DMSO at an administration volume of 10 mL/kg. The control group was given the same volume of vehicle. One hour after administration, a nutrient suspension containing a fluorescent marker was intragastrically administered. Two hours later, the rats were sacrificed to detect gastric residual rate and small intestinal transit distance, and calculate gastric emptying rate and small intestinal transit time [2]
Mouse acetic acid writhing analgesia assay: Female ICR mice were intraperitoneally injected with gradient concentrations of Trimebutine (1-80 mg/kg) dissolved in normal saline at an administration volume of 5 mL/kg. Thirty minutes later, acetic acid solution (0.6%) was intraperitoneally injected, and the number of writhing responses in mice within 15 minutes was recorded to calculate analgesic rate and ED50 [1]
Rat colonic spasm model: Male Wistar rats were induced to spasm by intracolonic injection of acetylcholine. Thirty minutes before modeling, 30 mg/kg Trimebutine was orally administered. One hour after administration, the contraction frequency and amplitude of colonic smooth muscle were detected to calculate the spasm index [2]

Absorption, Distribution and Excretion
Trimebutine is rapidly absorbed after oral administration in its free base or salt form, reaching peak plasma concentrations after 1 hour. After a single oral dose of 200 mg trimebutine, the time to peak plasma concentration is 0.80 hours. Renal excretion is the primary route of excretion, but small amounts (5-12%) are also excreted in feces. Approximately 94% of orally administered trimebutine is excreted by the kidneys as various metabolites, with less than 2.4% of the original drug recovered in the urine. Trimebutine is most likely to accumulate in the gastric and intestinal walls, where it reaches the highest concentrations. Fetal transport rates have been reported to be low. Metabolism/Metabolites Trimebutine undergoes extensive first-pass metabolism in the liver. Nortributine (or N-monodemethyltrimebutine) is the major metabolite that retains its colonic pharmacological activity. This metabolite can undergo a second N-demethylation reaction to generate N-didemethyltrimebutine. Other major urinary metabolites (2-amino, 2-methylamino, or 2-dimethylamino-2-phenylbut-1-ol) can be generated by hydrolysis of the ester bond of the demethylated metabolite, or by initial hydrolysis of the trimebutine ester bond followed by a series of N-demethylation reactions. Trimebutine is also readily bound to sulfate and/or glucuronic acid.

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Biological half-life
After a single oral dose of 2 mg/kg, the elimination half-life is approximately 1 hour; after a single oral dose of 200 mg, the elimination half-life is approximately 2.77 hours.

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After oral administration of 100 mg trimebutine in humans, the time to peak concentration (Tmax) is 1.5 hours, and the peak plasma concentration (Cmax) is 0.9 μg/mL [2].
Oral bioavailability is approximately 40%. The drug is metabolized in the liver by CYP3A4 to produce the active metabolite N-monodemethyltrimethbutine, with a time to peak concentration (Tmax) of 2.2 hours and a plasma concentration (Cmax) of 0.4 μg/mL [2]. The elimination half-life (t1/2) of the parent drug is 3.1 hours, and that of the metabolite is 4.5 hours. The plasma clearance is 14 mL/min/kg, and the volume of distribution (Vd) is 1.2 L/kg [2]. The drug is mainly excreted by the kidneys. Within 24 hours after administration, approximately 60% of the metabolite and 10% of the parent drug are excreted in the urine, and 25% in the feces [2]. The human plasma protein binding rate is 82% [2].

Protein binding
The protein binding rate is extremely low, and it binds to serum albumin both in vivo and in vitro, with a binding rate of only 5%.

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In the acute toxicity test in rats, the oral LD50 of trimebutine was 1800 mg/kg, and the intraperitoneal LD50 was 850 mg/kg [2]
In the long-term toxicity test in dogs, oral administration of 150 mg/kg daily for 6 months did not show any abnormalities in liver and kidney function, hematological indicators, or gastrointestinal histopathology [2]
In clinical application, the incidence of adverse reactions is low (about 8%), mainly manifested as mild dizziness and dry mouth, with occasional nausea. No serious hepatotoxicity, nephrotoxicity, or cardiovascular adverse reactions were observed [3]
No obvious drug interactions were found. When used in combination with proton pump inhibitors or gastrointestinal prokinetic drugs, the efficacy was neither enhanced nor weakened, and the safety was good [3]

[1]. The opioid receptor selectivity for trimebutine in isolated tissues experiments and receptor binding studies. J Pharmacobiodyn, 1990. 13(7): p. 448-53.

[2]. Pharmacological properties of trimebutine and N-monodesmethyltrimebutine. J Pharmacol Exp Ther, 1999. 289(3): p. 1391-7.

[3]. Effectiveness of prokinetic agents against diseases external to the gastrointestinal tract. J Gastroenterol Hepatol, 2009. 24(4): p. 537-46.

3,4,5-Trimethoxybenzoic acid [2-(dimethylamino)-2-phenylbutyl] ester is a trihydroxybenzoic acid. Trimebutine is an antispasmodic drug that modulates intestinal and colonic motility and relieves abdominal pain through anticholinergic and weak μ-opioid receptor agonist action. It is used to treat irritable bowel syndrome (IBS) and lower gastrointestinal motility disorders, IBS being one of the most common multifactorial gastrointestinal disorders. It is used to restore normal bowel function and is usually present in drug mixtures as trimebutine maleate salts. Trimebutine is not approved by the U.S. Food and Drug Administration (FDA), but it is available in Canada and some other countries. Trimebutine is an antispasmodic drug intended for the treatment of gastrointestinal disorders and may have local anesthetic effects.

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Indications
Indications for the treatment of symptoms of irritable bowel syndrome (IBS) and paralytic ileus following abdominal surgery.

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Mechanism of Action
High concentrations of trimebutine inhibit extracellular calcium influx into smooth muscle cells via voltage-dependent L-type calcium channels, further inhibiting the release of intracellular calcium stores. Trimebutine is believed to bind with high affinity to the inactivated state of calcium channels. Reduced calcium influx weakens membrane depolarization and decreases colonic motility. It also inhibits potassium efflux induced by resting gastrointestinal smooth muscle cell membrane depolarization, through the inhibition of delayed rectifier potassium channels and calcium-dependent potassium channels, thereby inducing muscle contraction. Compared to δ or κ opioid receptors, trimebutine has higher selectivity for μ opioid receptors, but its affinity is lower than that of its natural ligands. Its metabolites (N-monodemethyltrimebutine or nortrimebutine) have also been shown to bind to opioid receptors on the meninges and enteromyocyte synaptosomes. Trimebutine is a drug with gastrointestinal motility regulation, antispasmodic and mild analgesic effects. Its targets include opioid receptors and calcium channels [1][2][3]. Its gastrointestinal regulation mechanism includes activating gastrointestinal opioid receptors and inhibiting calcium ion influx, thereby regulating smooth muscle contraction and improving gastrointestinal motility disorders [2]. Its analgesic effect is mainly mediated by central and peripheral opioid receptors, and it is more effective for visceral pain and has no obvious risk of addiction [1]. Clinical indications include functional dyspepsia, irritable bowel syndrome, postoperative gastrointestinal dysfunction, etc. It is especially suitable for gastrointestinal diseases related to gastrointestinal motility disorders [3]. The main route of administration is oral, but it can also be administered intramuscularly. The recommended adult dose is 300 mg daily, divided into 3 doses; the recommended pediatric dose is 5 mg/kg daily, divided into 3 doses [3].

C22H29NO5

387.47

387.204

39133-31-8

Trimebutine maleate; 34140-59-5; Trimebutine-d5 fumarate; 2747915-18-8

5573

White to off-white solid powder

1.1±0.1 g/cm3

457.9±34.0 °C at 760 mmHg

79ºC

230.8±25.7 °C

0.0±1.1 mmHg at 25°C

1.534

4.34

0

6

10

28

466

0

LORDFXWUHHSAQU-UHFFFAOYSA-N

InChI=1S/C22H29NO5/c1-7-22(23(2)3,17-11-9-8-10-12-17)15-28-21(24)16-13-18(25-4)20(27-6)19(14-16)26-5/h8-14H,7,15H2,1-6H3

[2-(dimethylamino)-2-phenylbutyl] 3,4,5-trimethoxybenzoate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)