5-HT3 Receptor (IC50 = 0.33 nM)

In cultured rat cortical cells, hydrogen peroxide-induced neurotoxicity is lessened when 5-HT3 receptors are blocked with Bemisetron (MDL7222). At 1 μM and 5 μM, Bemisetron (0.01, 0.1, and 1 μM, 15 hours) and Y25130 (0.05, 0.5, and 5 μM) reduced H2O2-induced MTT reduction by 74.9±2.4 and 79.0 ±2.5%, respectively, concentration-dependently. are the maximum effects, in that order [2]. Bemisetron (1 μM), Y25130 (5 μM), or MK-801 (10 μM) pretreatment for 20 minutes considerably (but not totally) reduces the H2O2-induced rise in [Ca2+]c [2]. Significant blocking of the H2O2-induced rise in caspase-3 immunoreactivity can be achieved with bemisetron (1 μM, 15 hours) [2].

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The present study was performed to examine the neuroprotective effects of 5-hydroxytryptamine (5-HT)(3) receptor antagonists against hydrogen peroxide (H(2)O(2))-induced neurotoxicity using cultured rat cortical neurons. Pretreatment of 5-HT(3) receptor antagonists, tropanyl-3,5-dichlorobenzoate (MDL72222, 0.1 and 1 microM) and N-(1-azabicyclo[2.2.2.]oct-3-yl)-6-chloro-4-ethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-8-carboxamide hydrochloride (Y25130, 0.5 and 5 microM), significantly inhibited the H(2)O(2) (100 microM)-induced neuronal cell death as assessed by a MTT assay and the number of apoptotic nuclei, evidenced by Hoechst 33342 staining. The protective effects of MDL72222 (1 microM) and Y25130 (5 microM) were completely blocked by the simultaneous treatment with 100 microM 1-phenylbiguanide, a 5-HT(3) receptor agonist, indicating that the protective effects of these compounds were due to 5-HT(3) receptor blockade. In addition, MDL72222 (1 microM) and Y25130 (5 microM) inhibited the H(2)O(2) (100 microM)-induced elevation of cytosolic Ca(2+) concentration ([Ca(2+)](c)) and glutamate release, generation of reactive oxygen species (ROS), and caspase-3 activity. These results suggest that the activation of the 5-HT(3) receptor may be partially involved in H(2)O(2)-induced neurotoxicity, by membrane depolarization for Ca(2+) influx. Therefore, the blockade of 5-HT(3) receptor with MDL72222 and Y25130 may ameliorate the H(2)O(2)-induced neurotoxicity by interfering with the increase of [Ca(2+)](c), and then by inhibiting glutamate release, generation of ROS and caspase-3 activity[2].

Male adult albino mice were given Bemelsetron intraperitoneally at doses of 0.1, 1, and 10 mg/kg. There were no notable changes in catalepsy with the lowest dose. On the other hand, 10 mg/kg considerably increased this occurrence (from 60 minutes after haloperidol), while bemetesetron (1 mg/kg) dramatically decreased catalepsy (from 90 minutes after haloperidol). ). After administering haloperidol, the largest potentiation effect (about 4.5 times the control value) happens at 60 minutes and the maximum inhibitory effect (around 75%) on catalepsy occurs 120 minutes later [3].

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Typical neuroleptics (e.g. haloperidol) can induce a cataleptic state in rodents by means of striatal DA receptor blockade. It has been shown that drugs which influence central serotonergic (5-HTergic) mechanisms can modify neuroleptic-induced catalepsy, suggesting that dopaminergic transmission is under 5-HTergic modulation. The aim of this study was to examine the effects of bemesetron and granisetron, two selective 5-HT3 receptor antagonists, on this catalepsy in mice. Catalepsy was induced with haloperidol (1.5 mg/kg, i.p.) and measured at 30-min intervals by means of a bar test. Drugs (or saline, for the controls) were injected i.p. 20 min before haloperidol, with each animal used only once. Bemesetron significantly reduced catalepsy at a dose of 1 mg/kg, whilst 10 mg/kg potentiated the phenomenon and 0.1 mg/kg was found to be without effect. Granisetron inhibited catalepsy at doses of 0.04 and 0.1 mg/kg while 4 mg/kg of the antagonist significantly increased the duration of catalepsy. These data suggest that 5-HT3 receptors play a role in neuroleptic-induced catalepsy. Considering the high affinities of both antagonists for 5-HT3 receptors, it is tempting to speculate that the potentiation of catalepsy by high doses of them is due to non 5-HT3 receptor mechanisms[3].

Cell viability assay [2]
Cell Types: Primary cortical neurons Cell
Tested Concentrations: 0.01-1 μM
Incubation Duration: 20 minutes (pre-treatment); 15 hrs (hours) (after incubation)
Experimental Results: Concentration-dependent reduction of H2O2-induced MTT reduction reduction, showing 74.9±2.4% at 1 μM, which is the maximum effect.

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Western Blot Analysis[2]
Cell Types: Primary Cortical Neurons Cell
Tested Concentrations: 1 μM
Incubation Duration: 15 hrs (hours)
Experimental Results: Dramatically blocked H2O2-induced increase in caspase-3 immunoreactivity.

Animal/Disease Models: Male adult albino mouse, weight 26-36 g[3]
Doses: 0.1-10 mg/kg
Route of Administration: intraperitoneal (ip) injection, 20 minutes (pretreatment) + 180 minutes (treatment)
Experimental Results: obvious at dose 1 Reduces catalepsy symptoms mg/kg, while 10 mg/kg enhances this phenomenon, while 0.1 mg/kg has no effect.

The intravenous LD50 in rats was 14 mg/kg (US Patent No. 4563465); the oral LD50 in mice was 90 mg/kg (US Patent No. 4563465); the intraperitoneal LD50 in mice was 32 mg/kg (US Patent No. 4563465); and the intravenous LD50 in mice was 24 mg/kg (US Patent No. 4563465).

[1]. Peters JA, et al. An electrophysiological investigation of the properties of 5-HT3 receptors of rabbit nodose ganglion neurones in culture. Br J Pharmacol. 1993 Oct;110(2):665-76.
[2]. Lee HJ, et al. Blockade of 5-HT(3) receptor with MDL7222 and Y25130 reduces hydrogen peroxide-induced neurotoxicity in cultured rat cortical cells. Life Sci. 2005 Dec 5;78(3):294-300.
[3]. Silva SR, et al. Effects of 5-HT3 receptor antagonists on neuroleptic-induced catalepsy in mice. Neuropharmacology. 1995 Jan;34(1):97-9.

1. This study used whole-cell and lateral patch-clamp recording modes to determine the biophysical and pharmacological properties of 5-hydroxytryptamine (5-HT)-induced currents in cultured rabbit nodal ganglion neurons. 2. In the studied cells, 49% of cells induced inward currents after perfusion with 10⁻⁵ M 5-HT at a negative clamp potential. The whole-cell response to 5-HT reversed at approximately -2 mV (E5-HT) and exhibited inward rectifying characteristics. 3. The effects of different ion substitutions on E5-HT indicated that the 5-HT-induced current was primarily mediated by the conductance of mixed Na⁺ and K⁺ cations, with a small or negligible contribution from Cl⁻ ions. Removal of Ca²⁺ and Mg²⁺ from the extracellular fluid enhanced the amplitude of the 5-HT-induced current. 4. In ex vivo patch-clamp experiments, perfusion of 10⁻⁶ M 5-HT induced a single-channel current with a string conductance of approximately 17 pS at -70 mV and an average slope conductance of 19 pS in the range of -100 to -40 mV. The 5-HT-induced single channel exhibited slight inward rectification characteristics, the frequency of which could be reduced by the 5-HT₃ receptor antagonist metoclopramide (10⁻⁶ M), but the amplitude remained unaffected. 5. Under -60 mV voltage clamping, whole-cell perfusion of 5-HT (3 × 10⁻⁷ to 3 × 10⁻⁵ M) produced a dose-dependent inward current, which could be mimicked by 2-methyl-5-HT and 1-phenylbiguanide, with equivalent molar ratios of 2.5 and 32 relative to 5-HT, respectively. 6. The whole-cell inward current induced by local application of 5-HT (10(-5) M) was not affected by 10(-6) M mesimergot, 10(-6) M ketoserin or 10(-6) M citalopram, but could be antagonized in a concentration-dependent manner by the selective 5-HT3 receptor antagonists topisetron (IC50 = 4.6 x 10(-11) M), ondansetron (IC50 = 5.7 x 10(-11) M) and bemistron (IC50 = 3.3 x 10(-10) M). Non-selective antagonists metoclopramide (IC50 = 1.2 x 10⁻⁸ M), cocaine (IC50 = 8.3 x 10⁻⁸ M), and (+)-tubocurarine (IC50 = 1.6 x 10⁻⁷ M) can also block the 5-HT response. [1]

C15H17CL2NO2

314.206

313.064

C, 57.34; H, 5.45; Cl, 22.56; N, 4.46; O, 10.18

40796-97-2

671690

White to off-white solid powder

1.344g/cm3

406.507ºC at 760 mmHg

199.648ºC

1.597

3.713

0

3

3

20

355

2

O=C(O[C@@H]1C[C@@H](N2C)CC[C@@H]2C1)C3=CC(Cl)=CC(Cl)=C3

MNJNPLVXBISNSX-PBWFPOADSA-N

InChI=1S/C15H17Cl2NO2/c1-18-12-2-3-13(18)8-14(7-12)20-15(19)9-4-10(16)6-11(17)5-9/h4-6,12-14H,2-3,7-8H2,1H3/t12-,13+,14?

[(1S,5R)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl] 3,5-dichlorobenzoate

MDL72222; MDL-72222; BEMESETRON; 40796-97-2; 3-Tropanyl-3,5-dichlorobenzoate; MDL 72222; MDL-72222; MDL 72,222; Bemesetron (MDL 72222); CHEMBL1365455; MDL 72222

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 : ~2 mg/mL (~6.37 mM)

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.)