5-HT4 Receptor

Tegaserod (3-5 μM; 24-72 hours) significantly increases apoptosis in a time- and dose-dependent manner [1]. Tegaserod (TM) exerts its anti-cancer effects independently of serotonin signaling. Tegaserod (TM) blunts of ribosomal protein S6 (S6) phosphorylation through the PI3K/Akt/mTOR pathway[1]. Tegaserod lowers p-S6 and p-p70 S6 (Thr421/Ser424)[1] at 3-5 μM over 8–18 hours. According to 5-HT2B receptor antagonist activity, tegaserod (0.1-3 μM; 24 hours) efficiently suppresses 5-HT-mediated contraction of the rat gastric fundus in vitro (pA2=8.3) [3].

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1 Tegaserod (Zelnorm) is a potent 5-hydroxytryptamine4 (5-HT4) receptor agonist with clinical efficacy in disorders associated with reduced gastrointestinal motility and transit. The present study investigated the interaction of tegaserod with 5-HT2 receptors, and compared its potency in this respect to its 5-HT4 receptor agonist activity. 2 Tegaserod had significant binding affinity for human recombinant 5-HT2A, 5-HT2B and 5-HT2C receptors (pKi=7.5, 8.4 and 7.0, respectively). The 5-HT2B receptor-binding affinity of tegaserod was identical to that at human recombinant 5-HT4(c) receptors (mean pKi=8.4) in human embryonic kidney-293 (HEK-293) cells stably transfected with the human 5-HT4(c) receptor. 3 Tegaserod (0.1-3 microm) inhibited 5-HT-mediated contraction of the rat isolated stomach fundus potently (pA2=8.3), consistent with 5-HT(2B) receptor antagonist activity. Tegaserod produced, with similar potency, an elevation of adenosine 3',5' cyclic monophosphate in HEK-293 cells stably transfected with the human 5-HT4(c) receptor (mean pEC50=8.6), as well as 5-HT4) receptor-mediated relaxation of the rat isolated oesophagus (mean pEC50=8.2) and contraction of the guinea-pig isolated colon (mean pEC50=8.3).[3]

Tegaserod (5 mg/kg/day; intraperitoneal injection; five days in a row) inhibits p-S6 in vivo, decreases metastasis, and delays the formation of tumors [1]. Tegaserod (0.1-2.0 mg/kg; i.p. 15 min before gastric loading) dramatically increases the rate at which gastric glucose is emptied from the stomach in db/db mice and maintains it at 0.1 mg/kg for 30 minutes. This results in an 80% reduction in the amount of food consumed[2].

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Gastric emptying of glucose was significantly slower in db/db mice than in control littermates. Tegaserod (0.1 mg kg(-1)) significantly accelerated the gastric emptying rate of glucose in db/db mice, reducing the fraction of the meal remaining in the stomach at 30 min by 80%. GR11308 blocked the gastrokinetic effects of tegaserod. Conclusions: Gastric emptying was impaired in db/db mice. Low dose tegaserod improved gastric emptying rates in this model of gastroparesis through the activation of 5-HT(4) receptors. These findings suggest that 5-HT(4) receptor agonists may prove useful for improving delayed gastric emptying in gastroparesis. [2]

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Following subcutaneous administration, Tegaserod (0.3 or 1 mg kg(-1)) inhibited contractions of the stomach fundus in anaesthetized rats in response to intravenous dosing of alpha-methyl 5-HT (0.03 mg kg(-1)) and BW 723C86 (0.3 mg kg(-1)), selective 5-HT2B receptor agonists. At similar doses, tegaserod (1 and 3 mg kg(-1) subcutaneously) evoked a 5-HT4 receptor-mediated increase in colonic transit in conscious guinea-pigs. 5 The data from this study indicate that tegaserod antagonizes 5-HT2B receptors at concentrations similar to those that activate 5-HT4 receptors. It remains to be determined whether this 5-HT2B receptor antagonist activity of tegaserod contributes to its clinical profile.[3]

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Tegaserod (TM) delays tumor growth, reduces metastases, increases survival and suppresses p-S6 in vivo. [1]
To evaluate the efficacy of TM against melanoma tumor growth we used a syngeneic immune-competent model. Mice were subcutaneously inoculated with B16F10 cells, and 7 days later, randomized and treated with daily injections of TM or vehicle for 5 days. Treatment significantly decreased tumor growth (Fig. 4a) and resulted in only slight decreases in weight following treatment (Additional file 1: Figure S6A). There were no changes in liver damage markers AST, LDH and ALT (Additional file 1: Figure S6B). The in vitro TM-mediated PI3K/Akt/mTOR signaling inhibition was re-capitulated in vivo. When immunohistochemical staining of tumor tissue harvested 13 days post inoculation was performed for phosphorylation of S6 (Ser235/236), one third of control tumor slides were classified as having a high positive score. This is sharp contrast to tumors from TM treated mice where only one slide scored as having a high positive score (Fig. 4b). Images were scored for positive staining using the IHC profiler which employs an automated, unbiased approach to evaluate antibody staining in tissue sections. Furthermore, tumor lysates from TM treated mice had significantly lower Akt and S6 phosphorylation levels (Fig. 4c)[1].

Binding assay conditions [3]
Human recombinant 5-HT2A, 5-HT2B, 5-HT2C and 5-HT4(c) receptor membrane radioligand-binding assays were conducted as described previously (Grossman et al., 1993; Stam et al., 1994; Bonhaus et al., 1995; Pindon et al., 2002). Briefly, membranes prepared from cells stably transfected with human recombinant 5-HT2A, 5-HT2B, 5-HT2C and 5-HT4(c) receptors were incubated with radiolabelled ligands with a high affinity for the given receptor, that is, [3H]ketanserin, [N-methyl-3H]lysergic acid diethylamide (LSD), [3H]mesulergine and [3H]GR113808, respectively. Nonspecific radioligand binding was defined by ketanserin (1 μM), 5-HT (10 μM), SB 242084 (10 μM) and GR113808 (1 μM), respectively.
Competition-binding studies were conducted with increasing concentrations of unlabelled ligand (10 pM–30 μM) and a fixed concentration of radioligand (in nM): [3H]ketanserin (0.5), [N-methyl-3H]LSD (1.2), [3H]mesulergine and [3H]GR113808 (0.15). For [3H]GR113808, the radioligand concentration was ∼6-fold the KD value, but for all others the concentration was at, or close to, the KD. Following an incubation period sufficient to reach equilibrium: 15 min at 37°C, 30 min at 37°C, 30 min at 37°C and 60 min at 22°C, respectively, the membranes were harvested by rapid filtration and bound radioactivity quantitated by liquid scintillation spectroscopy.
Binding data were analysed by nonlinear regression analysis using GraphPad Prism™ software and a three-parameter model for one-site competition. pKi (negative decadic logarithm of Ki) values for test compounds were calculated from the best-fit IC50 values, and the Kd value of the radioligand, using the Cheng–Prusoff equation (Cheng & Prusoff, 1973): Ki=IC50/(1+[L]/Kd), where [L] is the concentration of the radioligand.

Apoptosis Analysis[1]
Cell Types: A375, RPMI-7951 (RPMI), SH4, B16F10, MeWo and MEL-JUSO
Tested Concentrations: 3, 5 μM
Incubation Duration: 24, 48, 72 h
Experimental Results: There was a significant time and dose-dependent increase in apoptosis in all cell lines.

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Western Blot Analysis[1]
Cell Types: RPMI, SH4 and B16F10 cells
Tested Concentrations: 3, 5 μM
Incubation Duration: 8 or 18 h
Experimental Results: diminished phosphorylation of the kinase directly upstream of S6, p70 S6 at Thr421/Ser424.

Animal/Disease Models: C57BL/6 J mice were subcutaneously (sc) injected with B16F10 cells[1]
Doses: 5 mg/ kg
Route of Administration: Administered intraperitoneally (ip) (ip) daily for five days
Experimental Results: Treatment Dramatically diminished tumor growth and resulted in only slight decreases in weight following treatment.

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Animal/Disease Models: Female C57BLKS/J db/db mice[2]
Doses: 0.1, 0.5 , 1.0, 2.0 mg/kg
Route of Administration: IP 15 min prior to gastric loading
Experimental Results: Produced a dramatic decrease in the fraction of the meal remaining in the stomach for doses as low as 0.1 mg/kg (0.1 mg/kg). Accelerated gastric emptying, with a reduction of nearly 80% in the fraction remaining at 30 min (P < 0.0001) (0.1 mg/kg). Induced a significant decrease in the gastric emptying rate as the amount of the meal remaining at 30 min was Dramatically greater (2.0 mg/kg). Resulted in inhibition of tegaserod-induced increased gastric emptying (0.1 mg/kg).

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\n\nC57BL/6 J mice were maintained under specific pathogen-free conditions. Seven to nine week old C57BL/6 J mice were subcutaneously injected with 5 × 105 B16F10 cells. Seven days post injection, when tumor volume reached approximately 50 mm3, mice were randomized and treated daily for 5 consecutive days with 5 mg/kg tegaserod or vehicle control (2.5% DMSO in PBS). Tegaserod and vehicle were administered intraperitoneally (i.p.). Tumors were measured using calipers and tumor volume was calculated using the following formula: (tumor length x width2)/2. For metastases quantification experiments, C57BL/6 J mice were intravenously injected with 2 × 105 B16F10 cells and treatment with Tegaserod and vehicle (administered i.p.) occurred 1 day post inoculation and continued three times weekly till day 14 post inoculation at which time mice were sacrificed. Metastases from lungs, stored in PBS for short term storage, were manually counted. For survival experiments, C57BL/6 J mice were intravenously injected with 105 B16F10 cells. Treatment with Tegaserod and vehicle (administered i.p.) occurred 1 day post inoculation and continued three times weekly till day 17 post inoculation. [1]

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\n\nGastric emptying rates of the glucose solution in control and diabetic mice were determined first. Mice were sacrificed 5, 10, 20, 30 and 40 min after the meal was instilled into the stomach. Four mice per data point were used. Serum glucose levels were checked using an Accu-chek III test reader (Roche Diagnostics Corporation, Indianapolis, IN, USA) with a drop of blood obtained by sectioning the tip of the tail of the animal.\n\nThe effect of tegaserod on gastric emptying was evaluated in diabetic mice. Tegaserod was given via an intraperitoneal (i.p.) injection 15 min prior to gastric loading at a dose of 0.1, 0.5, 1.0 or 2.0 mg kg−1. Mice were sacrificed 30 min after meal administration. Four db/db mice per dose of tegaserod were used. The effects of tegaserod were compared with those obtained after i.p. administration of vehicle (mixture of DMSO and saline).\n\nIn four mice, the effects of 5HT4 receptor blockade on tegaserod mediated effects were evaluated by administering GR11308, a 5HT4 antagonist (3.0 mg kg−1, i.p.) 10 min before tegaserod injection.\n\nDrug preparation Tegaserod was used. Injectable solutions of GR11308, a 5HT-4 antagonist, and tegaserod were prepared after solubilization in 1-methyl 2-pyrrolidinone. The solutions were diluted with normal saline to obtain final concentrations of tegaserod ranging from 0.01 to 0.2 mg ml−1, and a final concentration of GR11308 of 0.3 mg ml−1. For both compounds, the injectable volume was exactly 0.1 mL per 10 g of animal.[2]\n

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\n\nRats were allowed at least 30 min to stabilize following surgery. Typically, spontaneous rhythmical changes in balloon pressure commenced during this period, representing contractility of the stomach fundus. The selective 5-HT2B receptor agonists, α-methyl 5-HT (0.03 mg kg−1) and BW 723C86 (0.3 mg kg−1), or their vehicles, were administered via the jugular venous catheter (1 ml kg−1). These doses of α-methyl 5-HT and BW 723C86 were selected, as in initial experiments they were associated with increases in stomach pressure without marked changes in blood pressure. At 15 min after dosing with α-methyl 5-HT (0.03 mg kg−1), tegaserod or its vehicle was administered subcutaneously (1 ml kg−1). The selective 5-HT4 receptor antagonist, piboserod (1 mg kg−1; Sanger et al., 1998), was co-administered with tegaserod to exclude any influence of 5-HT4 receptor activation on stomach pressure. After a further 15 min, rats were dosed with α-methyl 5-HT (0.03 mg kg−1). Responses to α-methyl 5-HT were compared, by measuring the amplitude and area of stomach contractions, and data were expressed for the second α-methyl 5-HT challenge as a percentage of the first (statistical significance at P<0.05 by ANOVA and Dunnett's post hoc test, comparing the tegaserod and vehicle-induced responses). To avoid tachyphylaxis, each rat was challenged only once with BW 723C86, 15 min after subcutaneous co-administration of piboserod (1 mg kg−1) with either tegaserod (1 mg kg−1) or its vehicle, and data were compared by unpaired Student's t-test, with statistical significance set at P<0.05. The effect of the selective 5-HT2B/2C receptor antagonist, SB 206553 (1 mg kg−1; Kennett et al., 1996), on the BW 723C86 responses was also investigated. [3]\n

Absorption, Distribution and Excretion
The absolute bioavailability of tegaserod in fasting subjects is approximately 10%. The median time to peak plasma concentration (Tmax) of tegaserod is approximately 1 hour (range 0.7 to 2 hours). However, when tegaserod is taken 30 minutes before consuming a high-fat, high-calorie meal (approximately 150 calories from protein, 250 calories from carbohydrates, and 500 calories from fat), the AUC decreases by 40% to 65%, Cmax decreases by approximately 20% to 40%, and the median Tmax is 0.7 hours. Furthermore, plasma concentrations are similar when tegaserod is taken within 30 minutes before or 2.5 hours after a meal. Approximately two-thirds of orally administered tegaserod is excreted unchanged in feces, and the remaining one-third is excreted as metabolites in urine. Although tegaserod is not approved for intravenous administration, intravenous administration studies have shown a steady-state mean volume of distribution of 368 ± 223 L.
Although tegaserod is not approved for intravenous administration, intravenous administration studies have shown a mean plasma clearance of 77 ± 15 L/h.
Metabolism/Metabolites

Tegaserod is ultimately metabolized via hydrolysis and direct glucuronidation. The substance is first hydrolyzed in the stomach. It then undergoes oxidation and conjugation reactions to produce the major circulating tegaserod metabolite in human plasma, the so-called M29 metabolite or 5-methoxyindole-3-carboxylic acid. However, in vitro studies have shown that this major circulating metabolite has negligible affinity for the 5-HT(4) receptor. Furthermore, all three guanidino nitrogen atoms of tegaserod can be directly N-glucuronidated to produce three isomers of N-glucuronide—the so-called M43.2, M43.8, and M45.3 metabolites.
Known human metabolites of tegaserod include 1-[(E)-(5-hydroxy-1H-indole-3-yl)methyleneamino]-2-pentylguanidine.

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Biological Half-Life
After oral administration of tegaserod, the mean terminal elimination half-life is 4.6 to 8.1 hours.

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Currently, there is no information regarding the clinical use of tegaserod during lactation. Due to the potential for serious adverse reactions in breastfed infants, alternative medications are recommended.
◉ Effects on Breastfed Infants
As of the revision date, no relevant published information was found.
◉ Effects on Lactation and Breast Milk
As of the revision date, no relevant published information was found.

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Protein Binding Rate
The protein binding rate of tegaserod is approximately 98%.

[1]. Repurposing the serotonin agonist Tegaserod as an anticancer agent in melanoma: molecular mechanisms and clinical implications. J Exp Clin Cancer Res. 2020 Feb 21;39(1):38.

[2]. The effects of tegaserod, a 5-HT receptor agonist, on gastric emptying in a murine model of diabetes mellitus. Neurogastroenterol Motil. 2005 Oct;17(5):738-43.

[3]. The 5-HT4 receptor agonist, tegaserod, is a potent 5-HT2B receptor antagonist in vitro and in vivo. Br J Pharmacol. 2004 Nov;143(5):549-60.

Pharmacodynamics
Generally, tegaserod is identified as an agonist of the 5-HT4 (5-HT(4)) receptor and an antagonist of the 5-HT(2B) receptor, but is expected to bind very little to the 5-HT(1) receptor and has little affinity for the 5-HT(3) or dopamine receptors. In clinical trials of tegaserod, a centrally analyzed electrocardiogram was performed on 4605 male and female patients with constipation-predominant irritable bowel syndrome (IBS-C) and other related motility disorders who received tegaserod 6 mg twice daily or placebo. The absolute QTcF interval did not exceed 480 ms in all subjects treated with tegaserod. A QTcF interval prolongation of 30 to 60 ms was observed in 7% of patients treated with tegaserod, compared to 8% in those treated with placebo. A QTcF interval prolongation exceeding 60 ms was observed in 0.3% and 0.2% of subjects, respectively. Ultimately, the effect of tegaserod on the QTcF interval was deemed clinically insignificant. Furthermore, studies have revealed the potential of tegaserod and its major metabolite (M29 metabolite) to increase platelet aggregation in vitro. In one in vitro study, when tegaserod concentrations reached 10 times the maximum recommended plasma concentration (Cmax), it significantly increased platelet aggregation in a concentration-dependent manner, with an increase of up to 74% (range 11% to 74%) compared to the control solvent (this effect was enhanced by various agonists). In another in vitro study, when the concentration of the M29 metabolite reached 0.6 times the M29 Cmax, it also showed an increase in platelet aggregation of 5% to 16% compared to the control solvent. The clinical significance of these in vitro platelet aggregation results remains unclear. Background: Melanoma, especially in advanced patients unresponsive to immunotherapy and kinase inhibitors, urgently requires new treatment options. Methods: Drug screening, IC50 determination, and synergistic effects were performed using the MTT assay. Apoptosis was assessed by flow cytometry using Annexin V and 7AAD staining. TUNEL staining was performed using immunocytochemistry. Phosphorylation changes of key molecules in the PI3K/Akt/mTOR and other related pathways were detected by Western blotting and immunocytochemistry. To evaluate the in vivo antitumor activity of tegaserod, syngeneic intravenous and subcutaneous melanoma xenograft models were used. Immunocytochemical staining was performed to detect the expression of active Caspase-3, cleaved Caspase-8, and p-S6 in tumors. Immune infiltration was assessed by flow cytometry. Results: We screened 770 pharmacologically active and/or FDA-approved drugs and found that tegaserod (Zelnorm, Zelmac) is a novel anticancer compound that induces apoptosis in mouse and human malignant melanoma cell lines. Tegaserod (TM) is a 5-hydroxytryptamine receptor 4 agonist (HTR4) used to treat irritable bowel syndrome (IBS). The anti-apoptotic induction effect of TM on melanoma cells is not related to the 5-hydroxytryptamine signaling pathway, but is attributed to the inhibition of the PI3K/Akt/mTOR signaling pathway. Specifically, TM inhibits S6 phosphorylation in BRAFV600E and BRAF wild-type (WT) melanoma cell lines. In an in vivo model of normal homologous immunity, TM reduces tumor growth and metastasis and prolongs survival. In vivo experiments show that TM also induces tumor cell apoptosis, inhibits the PI3K/Akt/mTOR signaling pathway, and reduces S6 phosphorylation levels. Furthermore, TM reduces the infiltration of immunosuppressive regulatory CD4+CD25+ T cells as well as FOXP3 and ROR-γt-positive CD4+ T cells. Importantly, TM synergizes with vemurafenib (a standard treatment for patients with advanced disease carrying the BRAFV600E mutation) and in BRAFV600E and BRAFWT melanoma cell lines, TM can have an additive or synergistic effect with cobimetinib, thereby inducing an anticancer effect. Conclusion: In summary, we have identified a drug with anti-melanoma activity both in vitro and in vivo, which is expected to be used in combination with vemurafenib and cobimetinib, two standard treatments, for the treatment of BRAFV600E and BRAFWT melanoma. [1]

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C57BLKS/J db/db transgenic mice are a diabetic model that has been shown to have delayed gastric emptying. We evaluated the gastric emptying rate of C57BLKS/J mice and investigated the effect of the novel selective 5-HT₄ receptor partial agonist tegaserod on gastric emptying. Methods: We measured the gastric emptying rate of a 20% glucose meal in 12-20 week old female db/db mice and their littermates. In another group of db/db mice, we examined the effect of intraperitoneal injection of tegaserod (0.1-2.0 mg kg⁻¹) on gastric transit. Pretreatment with the specific 5-HT₄ receptor antagonist GR11308 was used to validate the mechanism of action of tegaserod on gastric emptying. Results: The gastric emptying rate of glucose in db/db mice was significantly lower than that in their littermates. Tegaserod (0.1 mg kg⁻¹) significantly accelerated the rate of glucose emptying from the stomach in db/db mice, reducing the proportion of food residue in the stomach by 80% at 30 minutes. GR11308 blocked the gastric motility effect of tegaserod. Conclusion: Gastric emptying is impaired in db/db mice. Low-dose tegaserod improved gastric emptying rate in this gastroparesis model by activating 5-HT₄ receptors. These findings suggest that 5-HT₄ receptor agonists may help improve delayed gastric emptying caused by gastroparesis. [2] Tegaserod (Zelnorm) is a potent 5-HT4 (5-HT₄) receptor agonist with clinical efficacy for diseases associated with slowed gastrointestinal motility and transit. This study investigated the interaction of tegaserod with 5-HT2 receptors and compared its efficacy in this regard with its activity as a 5-HT4 receptor agonist. Tegaserod showed significant binding affinity for human recombinant 5-HT2A, 5-HT2B, and 5-HT2C receptors (pKi values of 7.5, 8.4, and 7.0, respectively). In human embryonic kidney 293 (HEK-293) cells stably transfected with human 5-HT4(c) receptors, the binding affinity of tegaserod to the 5-HT2B receptor was the same as that of the human recombinant 5-HT4(c) receptor (mean pKi = 8.4). Tegaserod (0.1–3 μM) effectively inhibited 5-HT-mediated contraction in isolated rat gastric fundus (pA2 = 8.3), consistent with the activity of 5-HT(2B) receptor antagonists. Tegaserod, with similar potency, induced an increase in adenosine 3',5'-cyclic phosphate (mean pEC50 = 8.6) in HEK-293 cells stably transfected with human 5-HT4(c) receptors, and caused 5-HT4 receptor-mediated relaxation in isolated rat esophagus (mean pEC50 = 8.2) and contraction in isolated guinea pig colon (mean pEC50 = 8.3). Subcutaneous injection of tegaserod (0.3 or 1 mg kg⁻¹) inhibited the contractile response of anesthetized rat gastric fundus to intravenous injection of α-methyl-5-HT (0.03 mg kg⁻¹) and BW 723C86 (0.3 mg kg⁻¹, a selective 5-HT2B receptor agonist). At similar doses, tegaserod (subcutaneous injection of 1 and 3 mg kg⁻¹) induced increased colonic transit in conscious guinea pigs, a process mediated by 5-HT4 receptors.⁵ The data from this study suggest that tegaserod antagonizes 5-HT2B receptors at concentrations similar to those activating 5-HT4 receptors. Whether this 5-HT2B receptor antagonistic activity of tegaserod contributes to its clinical efficacy remains to be determined. [3]

C16H23N5O

301.39

301.19

C, 63.76; H, 7.69; N, 23.24; O, 5.31

145158-71-0

Tegaserod maleate;189188-57-6

135409453

White to off-white solid powder

1.2±0.1 g/cm3

470.6±48.0 °C at 760 mmHg

180-183ºC

238.4±29.6 °C

0.0±1.2 mmHg at 25°C

1.597

2.91

3

3

8

22

385

0

CCCCCN=C(N)N/N=C/C1=CNC2=C1C=C(C=C2)OC

IKBKZGMPCYNSLU-RGVLZGJSSA-N

InChI=1S/C16H23N5O/c1-3-4-5-8-18-16(17)21-20-11-12-10-19-15-7-6-13(22-2)9-14(12)15/h6-7,9-11,19H,3-5,8H2,1-2H3,(H3,17,18,21)/b20-11+

1-[(E)-(5-methoxy-1H-indol-3-yl)methylideneamino]-2-pentylguanidine

Tegaserod maleate; CPD000471618; 189188-57-6; DTXSID50904761; HMS2051J10

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)

DMSO : ~50 mg/mL (~165.90 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (16.59 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 5 mg/mL (16.59 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 5 mg/mL (16.59 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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