A 3-styrylchromone converted from trimebutine 3D pharmacophore possesses dual suppressive effects on RAGE and TLR4 signaling pathways.

Receptor for advanced glycation end-products (RAGE) and Toll-like receptors (TLRs) are potential therapeutic targets in the treatment of acute and chronic inflammatory diseases. We previously reported that trimebutine, a spasmolytic drug, suppresses RAGE pro-inflammatory signaling pathway in macrophages. The aim of this study was to convert trimebutine to a new small molecule using in silico 3D pharmacophore similarity search, and dissect the mechanistic anti-inflammatory basis. Of note, a unique 3-styrylchromone (3SC), 7-methoxy-3-trimethoxy-SC (7M3TMSC), converted from trimebutine 3D pharmacophore potently suppressed both high mobility group box 1-RAGE and lipopolysaccharide-TLR4 signaling pathways in macrophage-like RAW264.7 cells. More importantly, 7M3TMSC inhibited the phosphorylation of extracellular signaling-regulated kinase 1 and 2 (ERK1/2) and downregulated the production of cytokines, such as interleukin-6. Furthermore, 3D pharmacophore-activity relationship analyses revealed that the hydrogen bond acceptors of the trimethoxy groups in a 3-styryl moiety and the 7-methoxy-group in a chromone moiety in this compound are significant in the dual anti-inflammatory activity. Thus, 7M3TMSC may provide an important scaffold for the development of a new type of anti-inflammatory dual effective drugs targeting RAGE/TLR4-ERK1/2 signaling.

Repositioning Trimebutine Maleate as a Cancer Treatment Targeting Ovarian Cancer Stem Cells.

The overall five-year survival rate for late-stage patients of ovarian cancer is below 29% due to disease recurrence and drug resistance. Cancer stem cells (CSCs) are known as a major contributor to drug resistance and recurrence. Accordingly, therapies targeting ovarian CSCs are needed to overcome the limitations of present treatments. This study evaluated the effect of trimebutine maleate (TM) targeting ovarian CSCs, using A2780-SP cells acquired by a sphere culture of A2780 epithelial ovarian cancer cells. TM is indicated as a gastrointestinal motility modulator and is known to as a peripheral opioid receptor agonist and a blocker for various channels. The GI50 of TM was approximately 0.4 µM in A2780-SP cells but over 100 µM in A2780 cells, demonstrating CSCs specific growth inhibition. TM induced G0/G1 arrest and increased the AV+/PI+ dead cell population in the A2780-SP samples. Furthermore, TM treatment significantly reduced tumor growth in A2780-SP xenograft mice. Voltage gated calcium channels (VGCC) and calcium-activated potassium channels (BKCa) were overexpressed on ovarian CSCs and targeted by TM; inhibition of both channels reduced A2780-SP cells viability. TM reduced stemness-related protein expression; this tendency was reproduced by the simultaneous inhibition of VGCC and BKCa compared to single channel inhibition. In addition, TM suppressed the Wnt/ß-catenin, Notch, and Hedgehog pathways which contribute to many CSCs characteristics. Specifically, further suppression of the Wnt/ß-catenin pathway by simultaneous inhibition of BKCa and VGCC is necessary for the effective and selective action of TM. Taken together, TM is a potential therapeutic drug for preventing ovarian cancer recurrence and drug resistance.

Trimebutine attenuates high mobility group box 1-receptor for advanced glycation end-products inflammatory signaling pathways.

We previously identified papaverine as an inhibitor of receptor for advanced glycation end-products (RAGE) and showed its suppressive effect on high mobility group box 1 (HMGB1)-mediated responses to inflammation. Here, we found trimebutine to be a 3D pharmacophore mimetics of papaverine. Trimebutine was revealed to have more potent suppressive effects on HMGB1-induced production of pro-inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-α in macrophage-like RAW264.7 cells and mouse bone marrow primarily differentiated macrophages than did papaverine. However, the inhibitory effect of trimebutine on the interaction of HMGB1 and RAGE was weaker than that of papaverine. Importantly, mechanism-of-action analyses revealed that trimebutine strongly inhibited the activation of RAGE downstream inflammatory signaling pathways, especially the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), which are mediator/effector kinases recruited to the intracellular domain of RAGE. Consequently, the activation of Jun amino terminal kinase, which is an important effector kinase for the up-regulation of pro-inflammatory cytokines, was inhibited. Taken together, these results suggest that trimebutine may exert its suppressive effect on the HMGB1-RAGE inflammatory signal pathways by strongly blocking the recruitment of ERK1/2 to the intracellular tail domain of RAGE in addition to its weak inhibition of the extracellular interaction of HMGB1 with RAGE. Thus, trimebutine may provide a unique scaffold for the development of novel dual inhibitors of RAGE for inflammatory diseases.

Green conventional and first-order derivative fluorimetry methods for determination of trimebutine and its degradation product (eudesmic acid). Emphasis on the solvent and pH effects on their emission spectral properties.

In this report, the fluorescence properties of the antimuscarinic drug trimebutine maleate (TRB) were fully studied and characterized. TRB exhibited intrinsic fluorescence that is greatly dependent on the local environmental factors including the solvent nature and the pH. Yet, its fluorescence was not significantly influenced by the existence of some surface active agents and polymer. The outcomes of this investigation verified that TRB fluorescence emission is intense in ethanol: 1.0 M aqueous acetic acid (9:1, v/v) with emission maxima at 357 nm and excitation maxima at 270 nm. Whereas, going towards higher pH causes fluorescence quenching. These conditions permitted ultrasensitive fluorimetric determination of TRB over the concentration range of 2.00-1500.0 ng/mL with a lower detection limit of 0.40ng/mL Application for the determination of TRB in tablets, ampoule and suspension was successfully achieved with %recoveries ranged between 98.21-100.17%. Furthermore, a first order derivative fluorimetric method was validated for resolving and simultaneous determination of TRB and its degradation product and impurity, eudesmic acid (EUA) making use of the pH-mediated fluorescence spectral shift of EUA. An ethanolic solution containing acetate buffer (pH 5.3) was used for this goal with excitation at 255 nm and measurement of the first order derivative peak amplitudes at respective zero-crossing points of 375 and 351 nm over the corresponding concentration ranges of 20.00-500.00 and 10.00-300.00 ng/mL for TRB and EUA, respectively. The two methods were assessed regarding greenness and eco-friendship by the National Environmental Methods Index and analytical eco-scale score approaches which confirmed their excellent greenness and safety.

End point determination of blending process for trimebutine tablets using principle component analysis (PCA) and partial least squares (PLS) regression.

This study showed near Infrared (NIR) and Raman spectroscopy with a multivariate calibration approach were very effective to determine blend uniformity end-point. A set of 36 trimebutine samples containing magnesium stearate, stearic acid, colloidal silicon oxide, talc as excipients (0.9%∼1.8%) was acquired from six positions during blending processing with U-type blender from 0 to 30 min. Principle component analysis (PCA) with NIR and Raman spectral data was used to confirm the end-point of blending. After 30 min, the scores of principle component (PC) 1 and principle component (PC) 2 for samples moved into one point, which clearly indicated the mixture of sample became homogenous. In addition, NIR and Raman spectroscopy has been applied to the quantitative analysis of 20 trimebutine samples containing 2∼40% in mixture granules, which divided into a calibration set of 15 samples and a prediction set of 5 samples for NIR spectral data. The standard error of calibration (SEC) and standard error of prediction (SEP) are 0.15% and 0.13%, respectively using NIR while SEC and SEP of 0.95% and 0.91% are obtained using Raman spectroscopy. The results showed the NIR and Raman spectroscopy with a multivariate calibration such as PCA and PLS provide the possibility of real time monitoring of homogeneity and content uniformity during blending process.

Development of a novel combination tablet containing trimebutine maleate and mosapride citrate for the treatment of functional dyspepsia.

To develop a novel combination tablet which contained 100 mg trimebutine maleate and 5 mg mosapride citrate (TMCT) for the treatment of functional dyspepsia, the wet granulation method was used to prepare TMCTs with various amounts of diluents and stabilizers. The levels of impurities, the stability and the dissolution of the TMCTs were investigated. The oral bioavailability of drugs in the TMCTs was then evaluated and compared to the simultaneous oral administration of trimebutine maleate-loaded and mosapride citrate-loaded commercial products in the beagle dog. Among the diluents tested, D-mannitol was selected, since the microcrystalline cellulose and lactose did not inhibit the production of drug impurities due to their hygroscopic properties and chemical interactions, respectively. Furthermore, succinic acid was selected as the stabilizer because it gave the lowest level of total drug impurities of the organic acids tested. The combination tablet of trimebutine maleate and mosapride citrate prepared with D-mannitol and succinic acid gave a total drug content higher than 95% and total impurities lower than 0.5% at 25°C/60% RH and 40°C/75% RH during a 6-month period, indicating that the tablets were stable for at least 6 months. Furthermore, this combination tablet showed a similar dissolution to the trimebutine maleate-loaded and mosapride citrate-loaded commercial products and gave insignificantly different absorption compared to these commercial products in beagle dogs. Thus, the combination tablet of trimebutine maleate and mosapride citrate prepared with D-mannitol and succinic acid would be a stable and effective oral pharmaceutical product for the treatment of functional dyspepsia.

Spectrophotometric and liquid chromatographic determination of trimebutine maleate in the presence of its degradation products.

Three methods are presented for the determination of trimebutine maleate (TM) in the presence of its degradation products. The first method was based on a high performance liquid chromatographic (HPLC) separation of TM from its degradation products using an ODS column at ambient temperature with a mobile phase consisting of acetonitrile-5 mM heptane sulfonic acid disodium salt (45:55, v/v, pH 4) with UV detection at 215 nm. The second method depends on using first derivative spectrophotometry (1D) by measurement of the amplitude at 252.2 nm. The third method depends on using first derivative of the ratio spectrophotometry (1DD) by measurement of the amplitude at 282.4 nm where a normalized spectrum of 3,4,5-trimethoxy benzoic acid is used as divisor. The proposed HPLC and 1D methods were used to investigate the kinetics of acidic and alkaline degradation processes. The pH-rate profile of degradation of TM in Britton-Robinson buffer solutions within the pH range 2-11.9 was studied.

Ion-pair formation of Bi(III)-iodide with some nitrogenous drugs and its application to pharmaceutical preparations.

A systematic spectrophotometric study on the ion-pair formation of Bi(III)-iodide with amineptine hydrochloride, piribedil and trimebutine maleate is carried out. The optimal experimental conditions pH, concentration of Bi(III) nitrate, potassium iodide; and the nature and amount of organic solvent have been studied. The ion pairs are soluble in 1,2-dichloroethane and the optimum pH range is 2.0-2.8. By application of the methods of Sommer and Job involving non-equimolar solutions, the conditional stability constant (log K') of the Bi(III) piridedil ion pair (1:1) at the optimum pH of 2.4 and an ionic strength (mu) 0.1 M, was found to be 5.436. The validity of Beer's law has been tested in the concentration range 5-50 microg ml(-1) in the organic layer, the relative standard deviation is less than 1%. The method is applied to the determination of these drugs in tablets without interference.