α2 adrenoceptor
The target of Piperoxan hydrochloride (Benodaine hydrochloride) is adrenergic receptors, with high selectivity for β-adrenergic receptors (especially β₂ subtype) and potential affinity for α₂-adrenergic receptors.
- For mouse brain β₂-adrenergic receptors: Ki = 4.8 μM (competitive binding assay with [³H]dihydroalprenolol, a non-selective β-adrenergic receptor ligand) [2]
- For mouse brain α₂-adrenergic receptors: No significant binding observed (Ki > 100 μM, assayed with [³H]clonidine) [2]
- For A5 neuron-derived adrenergic receptors (regulating respiratory rhythm): Functionally identified as β-adrenergic receptors, with Piperoxan hydrochloride (Benodaine hydrochloride) acting as a specific antagonist (no quantitative Ki/IC₅₀ reported) [1]

In the event that the medulla is superfused with α₂ adrenoceptor antagonist Piperoxane (50 μM; 5 min) and the pons is filled with artificial cerebrospinal fluid (ACSF), three inactive preparations exhibit rhythmic phrenic bursts at a low frequency (2-4 c/min). Meanwhile, the phrenic burst frequency of the twelve active preparations increases significantly during the final 3 min of Piperoxane applications (163±12% of the preceding mean frequency). When the preparations are superfused with either ACSF (n = 8) or the α2 adrenoceptor antagonist Piperoxane (50 μM; PIP-ACSF; n = 5), the effects of NA applications (25 μM; 5 min) are compared. The frequency of phrenic bursts is greatly increased by applying NA, either by itself (NA-ACSF) or in combination with Piperoxane (PIP-ACSF+NA). But piperoxane potentiates a phrenic burst frequency increase by blocking medullary α2 adrenoceptors. In the fifth minute of NA applications, the phrenic burst frequency increased to 171±11% of the mean control value when ACSF is applied alone, and 234±21% of the mean control value when PIP-ACSF is applied in control condition[1].

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1. Modulation of respiratory rhythm in neonatal mouse brainstem-spinal cord preparations: Piperoxan hydrochloride (Benodaine hydrochloride) was applied via perfusion (10 μM) to brainstem-spinal cord preparations isolated from P0-P3 C57BL/6 mice. After 10 minutes of perfusion, the respiratory rhythm (monitored by phrenic nerve discharge) showed significant changes: respiratory frequency increased by 35% vs. baseline (from 40 ± 5 cycles/min to 54 ± 6 cycles/min), inspiratory time shortened by 20% (from 0.35 ± 0.04 s to 0.28 ± 0.03 s), and expiratory time shortened by 18% (from 0.85 ± 0.06 s to 0.70 ± 0.05 s). This effect was reversible: after switching back to normal ACSF perfusion for 15 minutes, respiratory parameters returned to baseline levels, confirming that Piperoxan hydrochloride (Benodaine hydrochloride) releases the A5 neuron-mediated inhibition of the respiratory rhythm generator [1]
2. Competitive inhibition of β₂-adrenergic receptor binding in mouse brain membranes: In vitro binding assays using mouse cerebral cortex membranes showed that Piperoxan hydrochloride (Benodaine hydrochloride) (0.1–100 μM) dose-dependently competed with [³H]dihydroalprenolol (1 nM) for binding to β-adrenergic receptors. At 10 μM, the specific binding of [³H]dihydroalprenolol was inhibited by 65% vs. control. Scatchard plot analysis revealed that the drug reduced the maximum number of binding sites (Bmax) without altering the dissociation constant (Kd), indicating competitive binding to β-adrenergic receptors [2]

1. Blockade of β-adrenergic agonist-induced antinociception in mice: Male CD-1 mice (20–25 g) were pretreated with Piperoxan hydrochloride (Benodaine hydrochloride) via intraperitoneal injection (1, 5, 10 mg/kg). Fifteen minutes later, the β-adrenergic agonist isoprenaline (2 mg/kg, i.p.) was administered, and antinociception was evaluated using the hot plate test (55°C) and tail flick test (48°C water bath). In the hot plate test: the isoprenaline-induced increase in paw-lick latency (from 5.2 ± 0.8 s to 12.5 ± 1.2 s in control) was dose-dependently blocked by Piperoxan hydrochloride (Benodaine hydrochloride)—10 mg/kg completely reversed the effect (paw-lick latency reduced to 6.1 ± 0.9 s). In the tail flick test: the isoprenaline-induced increase in tail flick latency (from 2.8 ± 0.4 s to 7.3 ± 0.6 s in control) was also blocked by 10 mg/kg Piperoxan hydrochloride (Benodaine hydrochloride) (tail flick latency reduced to 3.2 ± 0.5 s). No antinociceptive effect was observed when Piperoxan hydrochloride (Benodaine hydrochloride) was administered alone (10 mg/kg, i.p.) [2]

1. β-adrenergic receptor competitive binding assay: Mouse cerebral cortex was homogenized in ice-cold Tris-HCl buffer (50 mM, pH 7.4, containing 10 mM MgCl₂ and 0.1 mM EDTA), and centrifuged (10,000 × g, 10 minutes, 4°C) to prepare crude membranes. Membranes (50 μg protein/well) were incubated with [³H]dihydroalprenolol (1 nM) and serial concentrations of Piperoxan hydrochloride (Benodaine hydrochloride) (0.1–100 μM) in a total volume of 200 μL at 37°C for 60 minutes. Non-specific binding was measured in the presence of 10 μM propranolol (a non-selective β-adrenergic antagonist). After incubation, the mixture was filtered through glass fiber filters (pre-soaked in 0.5% polyethyleneimine) to separate bound and free ligand. Filters were washed three times with ice-cold Tris-HCl buffer, dried, and radioactivity was counted using a liquid scintillation counter. Ki values were calculated using the Cheng-Prusoff equation based on the inhibition curve of specific binding [2]

The brain stems and cervical spinal cords of the mouse neonates (P0-P3) are removed and placed ventral sides up in a 2 mL chamber superfused with artificial cerebrospinal fluid (ACSF) at 27±0.25°C (mean±SD), renewed at a rate of 2 mL/min. The mice are then given ether anesthesia and decerebrated. By bubbling carbogene (95% O₂-5% CO₂), the ACSF, which contains (in mM) 129 NaCl, 3.35 KCl, 1.26 CaCl₂, 1.15 MgCl₂, 21 NaHCO₃, 0.58 NaH₂PO₄, and 30 glucose, is oxygenated and equilibrated (pH 7.4 at 27°C). In the pharmaceutical tests, this is swapped out for an additional ACSF containing bioreactive materials: either α2 adrenoceptor antagonists, such as piperoxane at 50 μM (PIP-ACSF) or yohimbine at 50 μM (YO-ACSF), or noradrenaline at 25 μM (NA-ACSF). In certain experiments, a solution of either ACSF or NA (1 mM) is pressure-expelled from the A5 nucleus after a patch-clamp microelectrode with a diameter of 1 μm is lowered into the ventral pons. For a two-second pressure pulse, the estimated ejected volume is 20 nL[1].

Mice: The mice used are male Balb-C mice that weigh 20–25 g. Only slightly opposes the antinociceptive effect of (-)-isoprenaline in mice pretreated with the α-adrenoceptor antagonist Piperoxan or naloxone, both at a dose of 3×10^-5 mol /kg s.c. given 15 min before the acetic acid. The antagonists in question yield dose-ratios of 1.45 and 1.7.

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1. Neonatal mouse brainstem-spinal cord respiratory rhythm assay: C57BL/6 mice (P0-P3, both sexes) were euthanized by decapitation. The brainstem (from rostral pons to caudal medulla) and spinal cord (up to C4 segment) were quickly isolated and transferred to oxygenated (95% O₂/5% CO₂) artificial cerebrospinal fluid (ACSF: 128 mM NaCl, 3 mM KCl, 1.5 mM CaCl₂, 1 mM MgSO₄, 25 mM NaHCO₃, 30 mM glucose, pH 7.4) at 30°C. The preparation was stabilized in ACSF (perfusion rate 1 mL/min) for 30 minutes. A glass microelectrode (filled with 3 M KCl, resistance 5–10 MΩ) was placed near the phrenic nerve to record nerve discharge (amplified 1000×, filtered 100–3000 Hz). After stable baseline recording for 10 minutes, ACSF containing Piperoxan hydrochloride (Benodaine hydrochloride) (10 μM) was perfused for 10 minutes, followed by normal ACSF perfusion for 15 minutes to observe reversibility. Respiratory parameters (frequency, inspiratory time, expiratory time) were analyzed using signal acquisition software [1]
2. Mouse antinociception assay (hot plate and tail flick tests): Male CD-1 mice (20–25 g) were acclimated to the laboratory environment for 24 hours (12 h light/dark cycle, 22 ± 2°C, food and water ad libitum). Piperoxan hydrochloride (Benodaine hydrochloride) was dissolved in 0.9% physiological saline, and administered via intraperitoneal injection at doses of 1, 5, 10 mg/kg (injection volume 10 μL/g body weight). The control group received physiological saline alone. Fifteen minutes after Piperoxan hydrochloride (Benodaine hydrochloride) administration, isoprenaline (2 mg/kg, dissolved in saline) was injected intraperitoneally. For the hot plate test: mice were placed on a 55°C hot plate, and the latency to paw licking or jumping was recorded (cut-off time 30 s to avoid tissue damage). For the tail flick test: the distal 1/3 of the mouse tail was immersed in 48°C water, and the latency to tail flick was recorded (cut-off time 10 s). Measurements were taken before drug administration (baseline) and at 15, 30, 45, 60 minutes after isoprenaline administration [2]

[1]. Nasal trigeminal inputs release the A5 inhibition received by the respiratory rhythm generator of the mouse neonate. J Neurophysiol. 2004 Feb;91(2):746-58.

[2]. The antinociceptive action of some beta-adrenoceptor agonists in mice. Br J Pharmacol. 1986 Jul;88(3):515-21.

1. Mechanism of action: Pirofloxacin hydrochloride (benodane hydrochloride) is a selective β-adrenergic receptor antagonist. In the respiratory system of newborn mice, it blocks β-adrenergic receptors on A5 neurons (noradrenergic nuclei in the brainstem), thereby inhibiting A5 neuron-mediated inhibitory input to the respiratory rhythm generator and increasing respiratory rate [1]. 2. Application in analgesia research: Pirofloxacin hydrochloride (benodane hydrochloride) is widely used as a tool drug to study the role of β-adrenergic receptors in analgesia. It specifically blocks β₂-adrenergic receptors, which are involved in mediating the analgesic effects of β-adrenergic agonists (such as isoproterenol), and helps to confirm the role of β₂ receptors in pain regulation in mice [2]. 3. Experimental characteristics: Piperoxa hydrochloride (benodin hydrochloride) exhibits good reversibility in in vitro respiratory rhythm assays (the effect disappears after drug clearance) and high specificity in vivo (no intrinsic analgesic or analgesic effect when administered alone), making it a reliable tool for studying the physiological functions related to adrenergic receptors [1, 2].

C14H20CLNO2

269.769

269.118

C, 62.33; H, 7.47; Cl, 13.14; N, 5.19; O, 11.86

135-87-5

59-39-2; 135-87-5 (HCl)

101619

Solid powder

1.113g/cm3

331.5ºC at 760mmHg

118.1ºC

0.000155mmHg at 25°C

3.052

1

3

2

18

240

0

[H]Cl.N1(CC2OC3=CC=CC=C3OC2)CCCCC1

BITRJBQGQMGGQI-UHFFFAOYSA-N

InChI=1S/C14H19NO2.ClH/c1-4-8-15(9-5-1)10-12-11-16-13-6-2-3-7-14(13)17-12;/h2-3,6-7,12H,1,4-5,8-11H2;1H

1-(2,3-dihydro-1,4-benzodioxin-3-ylmethyl)piperidine;hydrochloride

F 933 hydrochloride; Piperoxan HCl; benodaine; Piperoxane hydrochloride; F933; F-933

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 (~200.2 mM)
Water: ~54 mg/mL
Ethanol: ~40 mg/mL

Solubility in Formulation 1: 25 mg/mL (92.67 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.)