Adult mouse skeletal muscle nicotinic acetylcholine receptor (nAChR, AChR) [2]

In TE671/RD cells, adiphenine (10 nM-1 mM; 3 minutes) inhibits α1*-nAChR function in a dose-dependent manner with an IC50 of 1.9 µM [1]. In SH-SY5Y cells, adiphenine (10 nM-1 mM; 3 minutes) inhibits α3α4*-nAChR function in a dose-dependent manner with an IC50 of 1.8 µM [1]. With an IC50 of 3.7 and 6.3 µM, respectively, adiphenine (10 nM-1 mM; 3 min) dose-dependently inhibits the function of α4β2- and α4β4-nAChR in SH-EP1 cells [1]. In HEK 293 cells, adiphenine (50–200 µM; 30–60 s) lowers the frequency of acetylcholine-induced single-channel currents [2].

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Adiphenine decreased the frequency of acetylcholine (ACh)-induced single-channel currents in adult mouse muscle AChR expressed in HEK293 cells. At a concentration of 60 µM, it reduced the frequency of opening events approximately 8-fold when activated by 0.5 µM ACh (a low concentration). [2]
Adiphenine significantly reduced the mean cluster duration (MCluD) of single-channel currents in a concentration-dependent manner. At 100 µM, it reduced cluster duration approximately 36-fold compared to control. [2]
Adiphenine (10 µM) caused a similar fractional decrease in cluster duration (~20%) across different ACh concentrations (20, 30, and 100 µM), indicating its main effect is not competitive antagonism at the agonist-binding site. [2]
Adiphenine did not significantly change the amplitude of single-channel currents. [2]
On macroscopic currents recorded from outside-out patches activated by 300 µM ACh, Adiphenine (up to 100 µM) had only a small effect on the extrapolated peak current (approximately 25% decrease at 100 µM). Its main effect was a concentration-dependent acceleration of the current decay (increased decay rate). The IC₅₀ for this effect on the decay time constant was 15 ± 3 µM (Hill coefficient = 1.18 ± 0.22). [2]
The acceleration of macroscopic current decay by Adiphenine was also observed at a lower ACh concentration (2 µM), where the decay time constant decreased from 211 ± 37 ms (control) to 117 ± 22 ms in the presence of 100 µM Adiphenine (applied simultaneously with ACh). [2]
Preincubation with Adiphenine was necessary to achieve its maximal effect on accelerating macroscopic current decay, suggesting a slow onset of action or the need to bind to the resting state before affecting the open state. [2]
Adiphenine stabilized the agonist-induced desensitized state(s) of the AChR, as evidenced by a significant reduction in the number of channel clusters observed after complete desensitization induced by a high ACh concentration (100 µM). [2]
The inhibition by Adiphenine was not significantly affected by the simultaneous presence of proadifen, suggesting different binding sites for the two drugs. [2]
The mutation αE262K in the mouse AChR significantly decreased the sensitivity of the receptor to Adiphenine. The decay time constants of macroscopic currents in the presence of 40 µM and 100 µM Adiphenine were slower for the αE262K mutant compared to wild-type. At the single-channel level, the magnitude of the decrease in mean cluster duration caused by 10 µM Adiphenine was smaller for the mutant (twofold) than for wild-type AChR (fivefold). This indicates residue αE262 is involved in the action of Adiphenine. [2]
The action of Adiphenine was only weakly voltage-dependent. With 100 µM Adiphenine, the decay time constant increased e-fold for a 230 mV depolarization. [2]
The mechanism of action is proposed to be primarily an acceleration of desensitization from the open state, rather than open-channel block. This is supported by the decrease in mean cluster duration and mean open time, the single-exponential decay of macroscopic currents, and the lack of a new closed component in single-channel records. [2]

In mice, adiphenine (ip) has an ED50 of 62 mg/kg and inhibits the hindleg tonic extensor component of maximal electroshock seizures (MES) [3].

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The study involved functional electrophysiological assays on the nicotinic acetylcholine receptor, which is a ligand-gated ion channel, not a classic enzyme. [2]

Human embryonic kidney (HEK) 293 cells were transfected with cDNAs encoding adult mouse skeletal muscle AChR subunits (wild-type α1, β1, δ, ε or mutant αE262K) using calcium phosphate precipitation at a ratio of 2:1:1:1 (α:β:δ:ε). Cells were used for electrophysiological recordings 1-2 days after transfection. [2]
Single-channel currents were recorded in the cell-attached or outside-out patch configurations. For cell-attached patches, the bath and pipette solutions contained specified ionic concentrations. ACh and Adiphenine were added to the pipette solution. Currents were recorded using a patch-clamp amplifier, digitized, and stored. Open-time, closed-time, and cluster-duration histograms were constructed and fitted with exponential functions using specialized software. Clusters were identified as series of openings separated by closed intervals longer than a critical duration (τ_crit) determined from histograms. [2]
For outside-out patch recordings (single-channel and macroscopic), the pipette and bath solutions had different specified compositions. Patches were excised and positioned at the outflow of a rapid perfusion system for solution exchange. For single-channel recordings, a three-tube perfusion system was used to apply solutions containing bath, ACh, or ACh plus Adiphenine. For macroscopic currents, a two-tube system was used. Different protocols were applied: 1) (+/-): constant exposure to drug-containing bath solution followed by a pulse of ACh; 2) (+/+): preincubation with drug-containing bath solution followed by a pulse of ACh plus the same drug; 3) (-/+): constant exposure to drug-free bath followed by a pulse of ACh plus drug. Macroscopic currents were filtered, digitized, and analyzed. The ensemble mean current was calculated from multiple traces and fitted with a single exponential function to obtain peak current and decay time constant. Inhibition curves were fitted to the Hill equation. [2]
To measure the kinetics of inhibition, a three-tube perfusion system was used with tubes containing bath solution, ACh, and ACh plus drug (or other combinations). Test pulses of ACh were applied, and the time course of current change upon drug introduction/removal was monitored and fitted with exponential functions. [2]

Absorption, Distribution and Excretion
This study describes the behavior of the dual-labeled drug adifenine in the rat brain. Macroscopic autoradiography revealed brain images at different time points post-injection. Several metabolites were identified at the brain tissue level, and the drug's crossing of the blood-brain barrier was compared with that of tritized water. The data provided very interesting clues about blood-brain distribution and brain tissue metabolism. High-performance liquid chromatography, macroscopic autoradiography, and tissue autoradiography can visualize drug fixation in brain tissue, thus revealing its mechanism of action. This study also investigated the distribution of dual-site labeled [14C]diethylethanolamine hydrochloride and [14C]diphenylacetic acid in rats and mice after intravenous injection and compared it with these. A biphasic decrease in blood radioactivity was observed. Bile excretion depends on the administered 14C-labeled compound: less than 5% was excreted from the diethylethanolamine moiety, while 100% was excreted from the carboxyl moiety. Less than 1% of the radioactivity observed in rat bile was associated with unmetabolized adefenine. …Shortly after administration, brain tissue uptake of [14C]-adefenine was 15 times higher than in the blood. Radioactivity was also found in the pituitary gland, adrenal glands, and melanin-like pigments at concentrations 30 times higher than in the blood. Metabolites/Metabolites: The major metabolites isolated from rat urine following a single administration of (14C)-adefenine or (3H)-adefenine were identified by chromatography and nuclear magnetic resonance spectroscopy, and compared with chemically synthesized standard reference compounds. Aldefenine is primarily metabolized via ester bond hydrolysis to diethylaminoethanol, diphenylacetic acid, diphenylacetic acid glucuronide, and small amounts of the corresponding glycine and glutamine conjugates. Following intravenous injection, researchers investigated the distribution of 14H-labeled adefenine at two sites in rats and mice… Preliminary metabolic studies identified three major metabolites: diphenylacetic acid, diethylethanolamine, and diphenylacetic acid glucuronide. Biological Half-Life: Male Wistar rats were intravenously injected with 15 μmol/kg of 3H-labeled adefenine. The concentrations of the parent drug in plasma and brain tissue were determined. Elimination of the 3H-labeled compound from plasma was monophasic, with a half-life of 13 minutes. The parent drug was still detectable in plasma within 30 minutes post-injection. The concentration changes of unmetabolized drug in the brain paralleled those in plasma, with half-lives ranging from 9 to 12 minutes. In all experiments, the concentrations of unmetabolized aldefenamine in the brain and plasma were highly correlated.

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Interactions
Although this drug has relatively low toxicity, it should not be used consecutively with morphine; concomitant use appears to cause anxiety and tachycardia. /Adefenine Hydrochloride/
In albino rats poisoned by anabacinth sulfate via gastric tube instillation of 346, 519, and 692 mg/kg, intramuscular injection of the antispasmodic drug (adefenine hydrochloride) and tropicamide was administered. The LD50 of anabacinth sulfate treated with adefenine hydrochloride was determined to be 570 mg/kg, and the LD50 of anabacinth sulfate treated with tropicamide was 403 mg/kg. The LD16 and LD84 values of adefenine hydrochloride and tropicamide were 460 and 712 mg/kg, and 288 and 498 mg/kg, respectively. Intramuscular injection of 20 mg/kg doses of adefenine hydrochloride and tropicamide reduced the LD50 of anabacinth sulfate by 271% and 191%, respectively. /Aldefenine Hydrochloride/

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Non-human Toxicity Values
Rat intravenous LD50: 27 mg/kg
Mice oral LD50: 600 mg/kg
Mice subcutaneous LD50: 400 mg/kg
Mice intravenous LD50: 21.5 mg/kg
For more complete non-human toxicity data for aldefenine (6 out of 6), please visit the HSDB record page.

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[1]. Local anesthetics noncompetitively inhibit function of four distinct nicotinic acetylcholine receptor subtypes. J Pharmacol Exp Ther. 2001 Dec;299(3):1038-48.

[2]. The local anaesthetics proadifen and adiphenine inhibit nicotinic receptors by different molecular mechanisms. Br J Pharmacol, 2009. 157(5): p. 804-17.

[3]. Anticonvulsant properties of procaine, cocaine, adiphenine and related structures. Proc Soc Exp Biol Med. 1955 Oct;90(1):192-5.

[4]. Adiphenine plasma levels and blood-brain barrier crossing in the rat. Eur J Drug Metab Pharmacokinet, 1985. 10(4): p. 273-8.

2,2-Diphenylacetic acid 2-(diethylamino)ethyl ester is a diarylmethane.

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Therapeutic Uses
Parasympathetic Nerve Blocker
Veterinary Drug: A smooth muscle relaxant used to treat spasms of the urinary system or gastrointestinal tract.
Adifenine has been used to relieve symptoms of gastrointestinal disorders characterized by spasms; gallbladder and bile duct spasms; dysmenorrhea; ureteral colic; and neurogenic bladder and certain other types of dysuria.
Difenine Hydrochloride
.../IT/ can relieve spasms of the gastrointestinal tract, biliary tract, ureter, and uterus without the effects of atropine-specific effects on salivary glands, sweat glands, gastric glands, eyes, or cardiovascular system, unless in large doses. Difenine Hydrochloride
For more complete data on the therapeutic uses of difenine (10 in total), please visit the HSDB record page.

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Drug Warning
Although this drug has relatively low toxicity, it should not be used consecutively with morphine; the combination appears to cause anxiety and tachycardia. Difening Hydrochloride

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Adiphenine is a local anesthetic closely related to proadifen and meproadifen. It is described as a non-competitive inhibitor (NCI) of the nicotinic acetylcholine receptor. [2]
The study demonstrates that despite being chemically similar to proadifen, Adiphenine inhibits the AChR via a different molecular mechanism and likely binds to a different site. Proadifen primarily acts on the resting state to induce a desensitized-like state, while Adiphenine accelerates desensitization from the open state and requires preincubation for full effect. [2]
The IC₅₀ value for its effect on macroscopic current decay (15 µM) is within the order of therapeutic concentrations attainable in blood for clinically used local anesthetics. [2]

347.165

50-42-0

2031

White to off-white solid powder

423ºC at 760 mmHg

71-74 °C(lit.)

133.2ºC

4.505

0

3

9

23

311

0

Cl[H].O(C(C([H])(C1C([H])=C([H])C([H])=C([H])C=1[H])C1C([H])=C([H])C([H])=C([H])C=1[H])=O)C([H])([H])C([H])([H])N(C([H])([H])C([H])([H])[H])C([H])([H])C([H])([H])[H]

JGOAIQNSOGZNBX-UHFFFAOYSA-N

InChI=1S/C20H25NO2/c1-3-21(4-2)15-16-23-20(22)19(17-11-7-5-8-12-17)18-13-9-6-10-14-18/h5-14,19H,3-4,15-16H2,1-2H3

2-(diethylamino)ethyl 2,2-diphenylacetate

Adiphenine hydrochloride NSC-129224 NSC 129224

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 : ≥ 100 mg/mL (~287.46 mM)
H2O : ≥ 50 mg/mL (~143.73 mM)

Solubility in Formulation 1: ≥ 3.25 mg/mL (9.34 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 32.5 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 3.25 mg/mL (9.34 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 32.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 3.25 mg/mL (9.34 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 32.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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