PTB-1B (IC50 = 1 μM); TC-PTP (IC50 = 224 μM)[1]
Protein tyrosine phosphatase 1B (PTP1B) (IC~50~ ≈ 1 μmol/l)
T-cell protein tyrosine phosphatase (TCPTP) (IC~50~ ≈ 224 μmol/l)
Dopamine transporter (DAT) (97% inhibition at screening concentration, but in vivo activity negated)
Norepinephrine transporter (NET) (89% inhibition at screening concentration, but in vivo activity negated) [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].

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Trodusquemine (10 μmol/l) significantly enhanced insulin-stimulated tyrosine phosphorylation of insulin receptor β (IRβ) in HepG2 cells, showing an approximately threefold increase compared to insulin treatment alone. Trodusquemine alone had no effect on IRβ phosphorylation. [1]
In an intact cell phosphatase activity assay using HepG2 cells, incubation with 10 μmol/l MSI-1436 resulted in a 53% inhibition of phosphatase activity compared to the control (no inhibitor). [1]
Screening assays indicated trodusquemine inhibited dopamine uptake and norepinephrine uptake in cellular assays, with reported IC~50~ values of 0.4 μmol/l and 0.7 μmol/l, respectively. However, subsequent in vivo studies did not confirm functional inhibition of these transporters. [1]
Trodusquemine showed no significant inhibition of dipeptidyl peptidase 4 (DPP-4), and no substantial binding to cannabinoid CB1, cholecystokinin (CCK), corticotropin-releasing factor (CRF), insulin, or leptin receptors. It also did not activate serotonin (5-HT~2C~) receptors. [1]

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]

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In diet-induced obese (DIO) AKR/J mice fed high-fat (45%) or very high-fat (60%) diets, intraperitoneal administration of trodusquemine (initial 10 mg/kg, followed by three weekly doses of 5 mg/kg) caused significant, sustained body weight loss proportional to pretreatment weight. This weight loss was fat-specific, with reduced total body fat and epididymal fat pad weight, while lean body mass was preserved. [1]
Trodusquemine treatment suppressed food intake in DIO mice. Unlike pair-fed controls whose weight loss plateaued, trodusquemine-treated mice continued to lose weight, suggesting the compound overrides compensatory metabolic responses to caloric restriction. [1]
Trodusquemine treatment significantly reduced plasma insulin and leptin levels in DIO mice compared to saline-treated controls within the same diet groups. [1]
In Sprague-Dawley rats, intraperitoneal administration of trodusquemine (10 mg/kg) enhanced insulin-stimulated tyrosine phosphorylation of hypothalamic IRβ (approximately twofold increase) and STAT3 (5.4-fold increase), indicating enhanced central insulin and leptin signaling via PTP1B inhibition. Trodusquemine alone increased hypothalamic STAT3 phosphorylation 2.7-fold. [1]
In vivo fast-scan cyclic voltammetry in rats and the tail suspension test in mice showed that systemic doses of MSI-1436 (10 mg/kg) did not alter dopamine reuptake in the nucleus accumbens, locomotor activity, or induce antidepressant-like effects, effectively ruling out dopamine or norepinephrine reuptake transporters as relevant in vivo targets. [1]

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]

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Inhibition of purified full-length PTP1B enzyme was measured. PTP1B and varying concentrations of trodusquemine (0-500 μmol/l) were incubated for 30 minutes in wells pre-coated with a tyrosine phosphopeptide substrate. Enzyme activity was quantified using an enzyme-linked immunosorbent assay, measuring absorbance at 405 nm. Trodusquemine potently inhibited PTP1B with an IC~50~ of approximately 1 μmol/l. [1]
Inhibition of T-cell protein tyrosine phosphatase (TCPTP) was assessed for selectivity. TCPTP and varying concentrations of trodusquemine (0-1000 μmol/l) were incubated with the substrate DiFMUP. The formation of the fluorescent product DiFMU was measured spectrofluorimetrically. Trodusquemine inhibited TCPTP with an IC~50~ of approximately 224 μmol/l, showing over 200-fold selectivity for PTP1B over TCPTP. [1]
Dipeptidyl peptidase 4 (DPP-4) inhibition was tested by pre-incubating the enzyme with 10 μmol/l trodusquemine, followed by addition of the substrate Ala-Pro-AFC. Formation of the fluorescent product AFC was measured spectrofluorimetrically. No inhibition was observed. [1]

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]

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To assess the effect on insulin signaling, HepG2 cells were serum-starved for 20 hours and then treated with 10 μmol/l trodusquemine in the presence or absence of 100 nmol/l insulin for 30 minutes. Cells were lysed, and proteins were immunoprecipitated with an IRβ antibody. Tyrosine phosphorylation of IRβ was analyzed by western blotting using a phospho-tyrosine antibody. [1]
An intact cell phosphatase activity assay was performed. HepG2 cells were pretreated with 10 μmol/l MSI-1436 or a control inhibitor for 10 minutes at 37°C. The cells were then incubated with the cell-permeable phosphatase substrate pNPP for 30 minutes at 37°C. The supernatant was collected, and the hydrolysis product pNP was measured spectrophotometrically at 405 nm to quantify phosphatase activity. [1]

In vivo voltammetry[1]
\nAdult 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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\n
\nTail suspension test[1]
\nMice (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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\n
\nGlucose tolerance test (GTT) and insulin tolerance test (ITT)[3]
\n3.5-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]
\nA 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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\n
\nFood intake[3]
\nMice 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).

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\nDiet-Induced Obesity (DIO) Mouse Study: Male AKR/J mice were fed 10%, 45%, or 60% fat diets ad libitum for approximately 14 weeks to induce obesity. Mice were then randomly assigned to treatment groups: trodusquemine (intraperitoneal injection, initial dose 10 mg/kg, followed by three weekly doses of 5 mg/kg), vehicle control (saline, 10 ml/kg, weekly 4x), or pair-fed control (saline injections, food matched to trodusquemine group intake). Body weight and food intake were monitored. After 23 days, mice were euthanized for blood and tissue collection. Body composition was analyzed by measuring moisture, fat (Soxhlet extraction), protein, and ash content. Epididymal fat pads were weighed. Plasma hormones (insulin, leptin, corticosterone) were measured by ELISA/RIA. Inguinal white adipose tissue was fixed, sectioned, stained with H&E, and adipocyte size was analyzed microscopically. [1]
\nRat Hypothalamic Tissue Collection for Signaling Analysis: Nine-week-old male Sprague-Dawley rats with ad libitum access to normal chow were dosed intraperitoneally with trodusquemine (10 mg/kg) or saline. After an overnight fast, rats received an intraperitoneal injection of saline or insulin (100 U/kg). Thirty minutes after the dose, animals were euthanized, hypothalami were harvested and homogenized in extraction buffer. Lysates were used for immunoprecipitation and western blot analysis of phosphorylated and total IRβ and STAT3. [1]
\nIn Vivo Voltammetry in Rats: Adult Sprague-Dawley rats were anesthetized and surgically implanted with cannulas and electrodes. After recovery, dopamine was electrically evoked in the nucleus accumbens every 5 minutes and measured using fast-scan cyclic voltammetry. Baseline dopamine dynamics were recorded before and after intraperitoneal injection of MSI-1436 (10 mg/kg), a known reuptake inhibitor (nonifensine, 7 mg/kg), or saline. Dopamine concentration and reuptake kinetics were monitored for 60 minutes. [1]
\nTail Suspension Test in Mice: Mice were administered MSI-1436 (5 or 10 mg/kg, IP), a positive control (desipramine, 20 mg/kg, IP), or vehicle. Thirty minutes later, mice were suspended by their tails in a controlled environment, and the duration of immobility during a 6-minute period was recorded by blinded observers. [1]

This study showed that trodusquemine can cross the blood-brain barrier and, after systemic administration, enhances insulin-stimulated hypothalamic IRβ tyrosine phosphorylation in vivo. [1] Previous studies cited in the article have shown that intraventricular injection of trodusquemine is 100 to 1000 times more effective at weight loss than parenteral administration, further supporting its ability to penetrate the central nervous system. [1] Note: The provided reference [1] does not include detailed pharmacokinetic parameters of trodusquemine, such as half-life, clearance, volume of distribution, or oral bioavailability. [1]

[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.

The disruption of endocannabinoid (eCB) signaling in the amygdala leads to stress-induced anxiety, but the mechanism remains unclear. eCB production is closely related to the function of the glutamate receptor mGluR5, which in turn depends on tyrosine phosphorylation. In this study, we discovered a novel pathway that regulates eCB-induced anxiety through mGluR5 phosphorylation. Mice lacking LMO4 (an endogenous inhibitor of the tyrosine phosphatase PTP1B) exhibited decreased mGluR5 phosphorylation, weakened eCB signaling, and severe anxiety; these symptoms could be reversed by genetic or pharmacological inhibition of PTP1B in the amygdala. Chronic stress mice exhibited elevated plasma corticosterone levels, decreased LMO4 palmitoylation, enhanced PTP1B activity, decreased amygdala eCB levels, and anxious behavior; these symptoms could be restored by inhibiting PTP1B or antagonizing glucocorticoid receptors. Corticosterone persistently reduces LMO4 palmitoylation levels in neurons and its inhibitory effect on PTP1B. These data collectively reveal a stress-responsive corticosterone-LMO4-PTP1B-mGluR5 cascade pathway that impairs amygdala endocannabinoid (eCB) signaling and promotes the development of anxiety. [2]

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Protein tyrosine phosphatase 1B (PTP1B) inhibits insulin signaling, interfering with its regulation 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 PTP1B inhibitor that can cross the blood-brain barrier to suppress food intake and promote insulin sensitivity and glycemic control. Trodusquemine is a naturally occurring cholesterol compound that can be purified from the liver of the squalus acanthias, but can also be prepared by a more cumbersome synthetic method that takes several weeks. This study tested a novel, readily synthesizable (only 2 days) polyaminosteroid derivative (Claramine) containing a spermidine group similar to Trodusquemine, and investigated its ability to inhibit PTP1B. Similar to Trodusquemine, Claramine selectively inhibits PTP1B but not its closest phosphatase, TC-PTP. In cultured neurons, both Claramine and Trodusquemine activated key components of the insulin signaling pathway and increased phosphorylation levels of insulin receptor β (IRβ), Akt, and GSK3β. Intraperitoneal injection of either Claramine or Trodusquemine effectively restored glycemic control in diabetic mice, as confirmed by glucose and insulin tolerance tests. A single intraperitoneal injection of Claramine, like an equivalent dose of Trodusquemine, suppressed food intake and led to weight loss without increasing energy expenditure. In conclusion, Claramine is a more readily synthesizable alternative compound for the treatment of type II diabetes. [3] Trodusquemine is an aminosterol that was originally isolated from shark tissue. It causes non-cachexia (fat-specific) weight loss, meaning it reduces adipose tissue without affecting lean muscle mass. [1] Its main mechanism of action for anti-obesity and insulin-sensitizing effects is selective allosteric, non-competitive inhibition of protein tyrosine phosphatase 1B (PTP1B), a negative regulator of insulin and leptin signaling pathways. This inhibition occurs both centrally (brain, particularly the hypothalamus) and peripherally. [1] By inhibiting hypothalamic PTP1B, trodusquemine enhances central insulin and leptin signaling, thereby reducing food intake and achieving sustained weight loss. Peripheral PTP1B inhibition enhances insulin sensitivity in tissues such as the liver. [1] This compound has shown potential as a treatment for obesity and type 2 diabetes and has been a focus of clinical trials. [1]

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.)