PTB-1B (IC50 = 1 μM); TC-PTP (IC50 = 224 μM)[1]

The inhibitory effect of MSI-1436 on TCPTP is weaker than its effect on PTP1B activity, with an IC50 value of 224 μM [1]. MSI-1436 (Trodusquemine, 10 μM) restores ERK phosphorylation of the mGluR1/5 agonist DHPG in F11 neuronal cells. MSI-1436 (10 uM) reverses DHPG-induced holding currents and restores DSI in LMO4KO BLA neurons [2]. MSI-1436 (0.1-100 μM) blocks PTP1B activity [3].

MSI-1436 (10 mg/kg, i.p.) causes mice to gain weight in response to obesity and decreases the amount of total body fat in addition to adipocyte size and lipid content in white adipose tissue [1]. Trodusquemine, or MSI-1436, reduces anxiety by reestablishing endocannabinoid (eCB) communication in the amygdala [2]. MSI-1436 (5 mg/kg, i.p.) inhibits food intake and causes weight reduction in CD1 mice. It also exhibits antidiabetic benefits in diabetic animals [3]. A single intraperitoneal dose of Claramine, like an equivalent dose of Trodusquemine, suppressed feeding and caused weight loss without increasing energy expenditure. In summary, Claramine is an alternative more easily manufactured compound for the treatment of type II diabetes.[3]

Quantitation of phosphatase activity was measured using an intact cell assay. Hep G2 cells were pretreated with 10 µmol/l MSI-1436 or sodium orthovanadate (100 µmol/l, postive control) for 10 min at 37 °C, then incubated with 10 mmol/l pNPP (a cell permeable hydrolyzable substrate) for 30 min at 37 °C. Samples of the supernatants were spectrophotometrically analyzed at OD405 for hydolyzed pNP, a direct end product of phosphatase activity.[1]

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Kinase assays[1]
Human kinases activities were measured using KINOMEscan. In brief, 256 DNA-tagged kinases, ligand affinity beads, and MSI-1436 (10 µmol/l) were incubated at room temperature, washed, and then eluted. Phage titer in the eluates was quantitated by real-time quantitative PCR.[1]

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Protein tyrosine phosphatase 1B (PTP1B) inhibits insulin signaling, interfering with its control of glucose homeostasis and metabolism. PTP1B activity is elevated in obesity and type 2 diabetes and is a major cause of insulin resistance. Trodusquemine (MSI-1436) is a "first-in-class" highly selective inhibitor of PTP1B that can cross the blood-brain barrier to suppress feeding and promote insulin sensitivity and glycemic control. Trodusquemine is a naturally occurring cholestane that can be purified from the liver of the dogfish shark, Squalus acanthias, but it can also be manufactured synthetically by a fairly laborious process that requires several weeks. Here, we tested a novel easily and rapidly (2 days) synthesized polyaminosteroid derivative (Claramine) containing a spermino group similar to Trodusquemine for its ability to inhibit PTP1B. Like Trodusquemine, Claramine displayed selective inhibition of PTP1B but not its closest related phosphatase TC-PTP. In cultured neuronal cells, Claramine and Trodusquemine both activated key components of insulin signaling, with increased phosphorylation of insulin receptor-β (IRβ), Akt and GSK3β. Intraperitoneal administration of Claramine or Trodusquemine effectively restored glycemic control in diabetic mice as determined by glucose and insulin tolerance tests. [3]

F11 neuronal cells (a chimeric cell line of the mouse neuroblastoma cell line N18TG-2 fused with embryonic rat dorsal-root ganglion neurons) can be easily cultured without special coating on the plates. F11 cells were grown and maintained as described previously.[3]

In vivo voltammetry[1]
Adult Sprague–Dawley rats (n = 12) were anesthetized (100 mg/kg ketamine, 50 mg/kg xylazine), cannulas placed in the nucleus accumbens core, and a stimulating electrode was placed in the ventral tegmental area. After recovery, dopamine was electrically evoked (stimulation train: 60 Hz, 24 p, 1.2 µA) every 5 min and measured using fast-scan cyclic voltammetry. Baseline dopamine was recorded before intraperitoneal injection of MSI-1436 (10 mg/kg), nomifensine (7 mg/kg), or saline. Peak dopamine concentration and time to decay to 37% (τ) were recorded over a 60-min period after dose and expressed as percent change from baseline. Fast-scan cyclic voltammetry was performed in both awake and behaving rats as described previously.

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Tail suspension test[1]
Mice (CD1, n = 6/group; Charles River Laboratories, Kingston, NY) were administered MSI-1436 (5 or 10 mg/kg, IP), norepinephrine reuptake inhibitor (positive control) (20 mg/kg desipramine HCl in 0.1% DMSO, IP), or vehicle. After 30 min, mice were suspended by their tail in a sensory controlled environment, and duration of immobility during a 6-min period was recorded by three independent observers masked to treatments.

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Glucose tolerance test (GTT) and insulin tolerance test (ITT)[3]
3.5-month-old male mice received intraperitoneal injection of Claramine or Trodusquemine dissolved in saline (5 mg/kg body weight) 24 h or 48 h prior to GTT or ITT, respectively. Mice were fasted overnight (∼16 h) with access to water prior to GTT at 10:00. Basal blood glucose sampled from the saphenous vein was measured using a standard glucometer prior to and after mice received a glucose bolus (2 g/kg body weight of 20% d-glucose) by intraperitoneal injection as described previously.[3]
A separate cohort of mice was fasted for 4 h prior to ITT, performed between 14:00 and 17:00. Human recombinant insulin (Sigma; Cat. #91077C), diluted in sterile saline was administered by intraperitoneal injection at 0.75 U/kg and blood glucose levels were monitored as above.[3]

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Food intake[3]
Mice were transferred to individual housing and habituated for 2 days. Food intake was measured after intraperitoneal injection of saline or Claramine or Trodusquemine (both at 5 mg/kg body weight).

[1]. Inhibition of PTP1B by trodusquemine (MSI-1436) causes fat-specific weight loss in diet-induced obese mice. Obesity (Silver Spring). 2010 Aug;18(8):1516-1523.

[2]. Chronic stress induces anxiety via an amygdalar intracellular cascade that impairs endocannabinoid signaling. Neuron. 2015 Mar 18;85(6):1319-31.

[3]. Functional properties of Claramine: a novel PTP1B inhibitor and mimetic compound. Biochem Biophys Res Commun. 2015 Feb 27;458(1):21-7.

Collapse of endocannabinoid (eCB) signaling in the amygdala contributes to stress-induced anxiety, but the mechanisms of this effect remain unclear. eCB production is tied to the function of the glutamate receptor mGluR5, itself dependent on tyrosine phosphorylation. Herein, we identify a novel pathway linking eCB regulation of anxiety through phosphorylation of mGluR5. Mice lacking LMO4, an endogenous inhibitor of the tyrosine phosphatase PTP1B, display reduced mGluR5 phosphorylation, eCB signaling, and profound anxiety that is reversed by genetic or pharmacological suppression of amygdalar PTP1B. Chronically stressed mice exhibited elevated plasma corticosterone, decreased LMO4 palmitoylation, elevated PTP1B activity, reduced amygdalar eCB levels, and anxiety behaviors that were restored by PTP1B inhibition or by glucocorticoid receptor antagonism. Consistently, corticosterone decreased palmitoylation of LMO4 and its inhibition of PTP1B in neuronal cells. Collectively, these data reveal a stress-responsive corticosterone-LMO4-PTP1B-mGluR5 cascade that impairs amygdalar eCB signaling and contributes to the development of anxiety.[2]

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Protein tyrosine phosphatase 1B (PTP1B) inhibits insulin signaling, interfering with its control of glucose homeostasis and metabolism. PTP1B activity is elevated in obesity and type 2 diabetes and is a major cause of insulin resistance. Trodusquemine (MSI-1436) is a "first-in-class" highly selective inhibitor of PTP1B that can cross the blood-brain barrier to suppress feeding and promote insulin sensitivity and glycemic control. Trodusquemine is a naturally occurring cholestane that can be purified from the liver of the dogfish shark, Squalus acanthias, but it can also be manufactured synthetically by a fairly laborious process that requires several weeks. Here, we tested a novel easily and rapidly (2 days) synthesized polyaminosteroid derivative (Claramine) containing a spermino group similar to Trodusquemine for its ability to inhibit PTP1B. Like Trodusquemine, Claramine displayed selective inhibition of PTP1B but not its closest related phosphatase TC-PTP. In cultured neuronal cells, Claramine and Trodusquemine both activated key components of insulin signaling, with increased phosphorylation of insulin receptor-β (IRβ), Akt and GSK3β. Intraperitoneal administration of Claramine or Trodusquemine effectively restored glycemic control in diabetic mice as determined by glucose and insulin tolerance tests. A single intraperitoneal dose of Claramine, like an equivalent dose of Trodusquemine, suppressed feeding and caused weight loss without increasing energy expenditure. In summary, Claramine is an alternative more easily manufactured compound for the treatment of type II diabetes.[3]

C40H78N4O8S

775.134331226349

774.55403

C, 57.84; H, 9.50; N, 5.86; O, 23.45; S, 3.36

1309370-86-2

MSI-1436;186139-09-3;MSI-1701;390808-64-7; 1309370-85-1 (phosphate)

145708148

White to off-white solid powder

6.24

8

12

21

53

1100

11

C(O)(C)C(=O)O.O[C@@H]1C[C@@]2([H])C[C@@H](NCCCNCCCCNCCCN)CC[C@]2(C)[C@@]2([H])CC[C@@]3([C@@]([H])([C@H](C)CC[C@H](C(C)C)OS(O)(=O)=O)CC[C@@]3([H])[C@]12[H])C

IVOWHCPDKHVRGQ-UHFFFAOYSA-N

InChI=1S/C37H72N4O5S.C3H6O3/c1-26(2)34(46-47(43,44)45)13-10-27(3)30-11-12-31-35-32(15-17-37(30,31)5)36(4)16-14-29(24-28(36)25-33(35)42)41-23-9-22-40-20-7-6-19-39-21-8-18-38;1-2(4)3(5)6/h26-35,39-42H,6-25,38H2,1-5H3,(H,43,44,45);2,4H,1H3,(H,5,6)

[6-[3-[3-[4-(3-aminopropylamino)butylamino]propylamino]-7-hydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]-2-methylheptan-3-yl] hydrogen sulfate;2-hydroxypropanoic acid

MSI-1436 lactate; MSI-1436lactate; 1309370-86-2; MSI-1436C; MSI1436C; MSI 1436C; Aminosterol 1436; Aminosterol1436; Aminosterol-1436

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~54 mg/mL (~53.99 mM)
0.1 M HCL : 50 mg/mL (~49.99 mM)

Solubility in Formulation 1: ≥ 3 mg/mL (3.00 mM) (saturation unknown) in 5% DMSO + 40% PEG300 +5% Tween-80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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