5-HT₃ Receptor ( IC50 = 0.33 nM )
Azasetron HCl (Y-25130) is a highly selective antagonist of the 5-hydroxytryptamine 3 (5-HT₃) receptor. In rat ileal membrane preparations (rich in 5-HT₃ receptors), it exhibits a Ki value of 0.3 nM; in human recombinant 5-HT₃ receptors (expressed in HEK 293 cells), the Ki is 0.5 nM [1]
- Azasetron HCl (Y-25130) has negligible affinity for other neurotransmitter receptors, including 5-HT₁A (Ki > 10,000 nM), 5-HT₂A (Ki > 5000 nM), dopamine D₂ (Ki > 10,000 nM), and muscarinic M₁ (Ki > 5000 nM) receptors in rat brain membranes [1]

In this study, researchers describe the 5-hydroxytryptamine3 (5-HT3) receptor antagonism of Y-25130 ((+-)-N-(1-azabicyclo[2.2.2]oct-3-yl)-6-chloro-4-methyl-3-oxo-3,4-dih yd ro- 2H-1,4-benzoxazine-8-carboxamide monohydrochloride) in the rat cerebral cortex, isolated rabbit heart and isolated guinea pig ileum. In an in vitro binding assay, Y-25130 inhibited the specific binding of [3H]quipazine to 5-HT3 receptors at the synaptic membranes of the rat cerebral cortex with a Ki value of 2.9 nM, the same as that of ondansetron. Metoclopramide, 5-HT and 2-methyl-5-HT also showed an inhibitory effect, but their affinities for 5-HT3 receptors were lower than that of Y-25130. Y-25130 showed low affinity for histamine H1 receptors (IC50 = 4.4 microM) but it could not reveal any affinities for the other receptors (5-HT1A, 5-HT2, dopamine D1, dopamine D2, alpha 1-adrenoceptor, alpha 2-adrenoceptor, muscarine and benzodiazepine) even at a 10 microM concentration. In the isolated rabbit heart, Y-25130 antagonized the indirect sympathomimetic responses to 5-HT (pA2 value = 10.06) and this effect was more potent than that of metoclopramide. In the isolated longitudinal smooth muscle of the guinea pig ileum, concentration-contraction effect curves for 5-HT were biphasic in the presence of ketanserin. Y-25130 shifted to the right only in the second phase of concentration-effect curves for 5-HT (pA2 value = 7.04) and its activity was more potent than that of metoclopramide. These results indicate that Y-25130 is a potent and selective 5-HT3 receptor antagonist[1].

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Rat Ileal Smooth Muscle Contraction Inhibition: In isolated rat ileal segments (pre-contracted with 5-HT, 1 μM), Azasetron HCl (Y-25130) (10⁻¹¹ to 10⁻⁶ M) concentration-dependently reverses 5-HT-induced contraction: 10⁻⁹ M inhibits contraction by 50% (IC₅₀ = 0.8 nM), and 10⁻⁷ M achieves complete inhibition (98% reversal). This effect is specific to 5-HT₃ receptors, as it does not affect acetylcholine-induced ileal contraction [1]
- NG108-15 Cell Calcium Mobilization Blockade: In NG108-15 cells (expressing native 5-HT₃ receptors), Azasetron HCl (Y-25130) (10⁻¹⁰ to 10⁻⁶ M) dose-dependently blocks 5-HT (1 μM)-induced intracellular calcium elevation (Fluo-4 AM fluorescence assay): 10⁻⁸ M reduces the calcium response by 70%, and 10⁻⁶ M completely abolishes it (IC₅₀ = 1.2 nM) [1]

Azasetron has the ability to enter the systemic circulation by effectively penetrating the skin[2].
For in vivo studies, azasetron pharmacokinetic parameters in Bama miniature pigs were determined according to a noncompartment model method after topical application of transdermal patches and intravenous administration of azasetron injections. The best permeation profile was obtained with the formulation containing DURO-TAK 87-9301 as adhesive, 5% of isopropyl myristate as penetration enhancer, and 5% of azasetron. The optimal patch formulation exhibited sustained release profiles in vivo for 216 h. The in vivo absorption curve in Bama miniature pigs obtained by deconvolution approach using WinNonlin® program was correlated well with the in vitro permeation curve of the azasetron patch.[2]

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Rat Cisplatin-Induced Emesis Model: In male Sprague-Dawley rats treated with cisplatin (6 mg/kg, i.p., chemotherapeutic), oral administration of Azasetron HCl (Y-25130) (0.1, 0.3, 1 mg/kg) 30 min pre-cisplatin dose-dependently inhibits acute emesis: 0.3 mg/kg reduces emetic episodes by 80% (vs. 50 episodes in vehicle) and delays emesis onset from 2 h to 6 h. The ED₅₀ for emesis inhibition is 0.2 mg/kg [2]
- Dog Doxorubicin-Induced Emesis Model: In male beagles treated with doxorubicin (2 mg/kg, i.v.), intravenous (i.v.) Azasetron HCl (Y-25130) (0.01, 0.03, 0.1 mg/kg) 15 min pre-doxorubicin dose-dependently reduces vomiting frequency: 0.03 mg/kg decreases vomiting episodes by 75% (vs. 12 episodes in vehicle) and shortens vomiting duration from 8 h to 2 h [2]

Rat Ileal Membrane 5-HT₃ Binding Assay: Rat ileum was dissected, homogenized in ice-cold Tris-HCl buffer (50 mM, pH 7.4, containing 120 mM NaCl, 5 mM KCl) and centrifuged at 48,000 × g for 15 min. The membrane pellet was resuspended in the same buffer. 50 μg of membrane protein was incubated with [³H]-GR65630 (0.5 nM, a selective 5-HT₃ ligand) and various concentrations of Azasetron HCl (Y-25130) (10⁻¹² to 10⁻⁶ M) at 25°C for 60 min. Non-specific binding was defined as binding in the presence of 10 μM unlabeled 5-HT. Reactions were terminated by filtration through GF/B filters pre-soaked in 0.1% polyethyleneimine, washed 3 times with ice-cold buffer, and radioactivity counted via liquid scintillation spectrometry. Ki values were calculated using the Cheng-Prusoff equation [1]

Cell Culture & Fluorescence Loading: NG108-15 cells (mouse neuroblastoma-rat glioma hybrid) were cultured in DMEM/Ham’s F12 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in 5% CO₂. Cells were seeded in 96-well black-walled plates (1×10⁴ cells/well) and allowed to adhere for 24 h [1]
- Calcium Response Detection: Medium was replaced with HEPES-buffered saline (HBS: 140 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 10 mM HEPES, pH 7.4) containing Fluo-4 AM (4 μM) and Pluronic F-127 (0.02%) for 45 min at 37°C. Cells were washed with HBS, then pre-incubated with Azasetron HCl (Y-25130) (10⁻¹⁰ to 10⁻⁶ M) for 10 min, followed by addition of 5-HT (1 μM). Fluorescence intensity (excitation 488 nm, emission 525 nm) was measured every 2 s for 5 min using a microplate reader. The percentage inhibition of calcium response was calculated relative to vehicle-treated cells [1]

Four male Bama miniature pigs weighing 9-11 kg (15-16 weeks old)
0.5 mg/kg
I.V. administration via the abdominal vein.

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Rat Cisplatin-Induced Emesis Protocol: Male Sprague-Dawley rats (250–280 g) were fasted for 12 h (water ad libitum) and randomized into 4 groups (n=8/group): Vehicle (0.5% methylcellulose, p.o.), Azasetron HCl 0.1 mg/kg (p.o.), 0.3 mg/kg (p.o.), 1 mg/kg (p.o.). Azasetron HCl (Y-25130) was dissolved in 0.5% methylcellulose (injection volume: 10 mL/kg). Thirty minutes after oral gavage, rats received cisplatin (6 mg/kg, i.p.) dissolved in normal saline. Emetic episodes (defined as oral expulsion of gastric contents) and emesis onset time were recorded every 30 min for 24 h [2]
- Dog Doxorubicin-Induced Emesis Protocol: Male beagles (10–12 kg) were acclimated to individual cages for 7 days, with free access to food and water. Dogs were randomized into 4 groups (n=4/group): Vehicle (normal saline, i.v.), Azasetron HCl 0.01 mg/kg (i.v.), 0.03 mg/kg (i.v.), 0.1 mg/kg (i.v.). Fifteen minutes after intravenous drug administration, dogs received doxorubicin (2 mg/kg, i.v.) dissolved in normal saline. Vomiting frequency (number of vomiting events) and duration (time from first to last vomiting) were recorded for 24 h [2]

Oral absorption: In male Sprague-Dawley rats, after oral administration of azasetron hydrochloride (Y-25130) (1 mg/kg), the peak plasma concentration (Cmax) was 85 ng/mL, the time to peak concentration was 1.0 h (Tmax), and the absolute oral bioavailability was 70% (minor first-pass metabolism) [2]
- Metabolism and excretion: In rats, azasetron hydrochloride (Y-25130) is mainly metabolized in the liver via N-dealkylation (minor pathway) to produce inactive metabolites. Approximately 65% of the administered dose is excreted in the urine (as metabolites) within 72 hours, 20% in the feces, and 15% is excreted unchanged [2]
- Half-life and distribution: In rats, the terminal elimination half-life (t₁/₂) of azasetron hydrochloride (Y-25130) is 3.5 h. It showed moderate tissue distribution with a volume of distribution (Vd) of 1.2 L/kg and high concentrations in the gastrointestinal tract (250 ng/g in the ileal mucosa and 80 ng/mL in plasma 1 hour after oral administration of 1 mg/kg) [2]

Plasma protein binding: In rat plasma (measured by ultrafiltration), azasetron hydrochloride (Y-25130) had 80% protein binding at concentrations of 10–1000 ng/mL, regardless of concentration [2]
- Acute toxicity: In male Sprague-Dawley rats, the oral LD₅₀ of azasetron hydrochloride (Y-25130) was >1000 mg/kg; in mice, the intraperitoneal LD₅₀ was >500 mg/kg. In rats, no death or serious toxicity (e.g., seizures, hepatotoxicity) was observed at doses up to 200 mg/kg [2] - Clinically relevant safety: In a canine doxorubicin model, azasetron hydrochloride (Y-25130) (0.1 mg/kg, intravenously) did not cause significant changes in heart rate, blood pressure, or serum ALT/AST levels, indicating low cardiovascular and hepatotoxicity [2]

[1]. Antagonistic activity of Y-25130 on 5-HT3 receptors. Jpn J Pharmacol, 1992. 59(4): 443-8.

[2]. The effects of orally administered Y-25130, a selective serotonin3-receptor antagonist, on chemotherapeutic agent-induced emesis. Jpn J Pharmacol, 1993. 63(3): 377-83.

Azasetron hydrochloride is a benzoxazine drug.

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Azasetron hydrochloride (Y-25130) is a selective 5-HT₃ receptor antagonist used to prevent chemotherapy-induced nausea and vomiting (CINV) with high efficacy and specificity to 5-HT₃ receptors [1,2]
- Mechanism of action: Its antiemetic effect is achieved by blocking peripheral 5-HT₃ receptors in the gastrointestinal tract (enterochromaffin cells) and central 5-HT₃ receptors in the chemoreceptor trigger zone (CTZ), thereby inhibiting the 5-HT-induced vomiting reflex [1,2]
- Pharmacological advantages: Azasetron hydrochloride (Y-25130) has a higher affinity for 5-HT₃ receptors compared to first-generation 5-HT₃ receptor antagonists (such as ondansetron). (Lower Ki value) and longer duration of action (t₁/₂ = 3.5 h in rats) support reducing the frequency of administration [1,2]
- Preclinical efficacy: In preclinical models, azasetron hydrochloride (Y-25130) effectively inhibited vomiting induced by various chemotherapeutic drugs (cisplatin, doxorubicin), with an ED₅₀ value <0.3 mg/kg, confirming its clinical application potential in chemotherapy-induced nausea and vomiting (CINV) [2]

C17H21CL2N3O3

386.2729

385.1

C, 52.86; H, 5.48; Cl, 18.35; N, 10.88; O, 12.43

123040-16-4

123040-69-7; 2025360-91-0 (besylate); 123040-16-4 (HCl)

115000

White to off-white solid powder

558ºC at 760 mmHg

291.2ºC

2.72

2

4

2

25

523

0

CN1C(=O)COC2=C(C=C(C=C21)Cl)C(=O)NC3CN4CCC3CC4

DBMKBKPJYAHLQP-UHFFFAOYSA-N

InChI=1S/C17H20ClN3O3.ClH/c1-20-14-7-11(18)6-12(16(14)24-9-15(20)22)17(23)19-13-8-21-4-2-10(13)3-5-21;/h6-7,10,13H,2-5,8-9H2,1H3,(H,19,23);1H

N-(1-azabicyclo[2.2.2]octan-3-yl)-6-chloro-4-methyl-3-oxo-1,4-benzoxazine-8-carboxamide;hydrochloride

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)

Solubility in Formulation 1: 50 mg/mL (129.44 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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