Voltage-gated Na⁺ channels, Ca²⁺ channels, K⁺ channels. [1]

In the plasma membrane of LM cells (transformed fibroblasts), prilocaine hydrochloride inhibits Na,K-ATPase more effectively at 37 °C (43.8 mM) than at 25 °C (28.2 mM) [2].

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Prilocaine caused concentration-dependent neurotoxic effects on NB2a cells, evaluated by neurite inhibition, cell viability, and apoptosis. The order of neurotoxicity (from most to least toxic) was tetracaine > prilocaine > lidocaine > procaine based on cell viability and apoptotic potency. [1]
- At 1 μl concentration, a statistically significant difference in cell viability was found between procaine and prilocaine groups (p < 0.05). At 10 μl, significant differences were observed between procaine and prilocaine groups (p < 0.01), as well as between lidocaine and prilocaine groups (p < 0.05). At 25 μl, no significant differences among groups. At 100 μl, no significant difference between prilocaine and other groups except procaine vs. tetracaine (p < 0.01). [1]
- For neurite inhibition (NI) and i-NOS (inducible nitric oxide synthase) immunostaining, no significant difference was detected between prilocaine and other groups except between procaine and tetracaine (p < 0.05 for NI; p < 0.01 for i-NOS). Prilocaine showed intermediate levels of NI and i-NOS staining compared to other anesthetics. [1]
- For TUNEL staining (apoptosis), significant differences were found between procaine and prilocaine groups (p < 0.01), as well as between lidocaine and tetracaine groups (p < 0.05). Prilocaine induced more apoptosis than procaine but less than tetracaine. [1]
- There was an inverse relationship between prilocaine concentration and cell viability. Prilocaine exerted its toxic effects by neurite inhibition at low concentrations and by apoptosis at high concentrations. [1]

Na,K-ATPase activity was measured using an enzyme-coupled method with an ATP-regenerating system. The assay mixture contained Heps buffer, NaCl, KCl, MgCl2, dithiothreitol, EDTA, phosphoenolpyruvate (PEP), NADH, lactate dehydrogenase, and pyruvate kinase. The reaction was initiated by adding Na2ATP, and the decrease in absorbance at 340 nm (due to NADH oxidation) was recorded. The millimolar absorptivity of NADH (6.22) was used to convert the rate of absorbance change to international units. For inhibition studies, the Na,K-ATPase-enriched membranes were preincubated with or without the anaesthetic for 20 minutes at the assay temperature (25°C or 37°C) before starting the reaction. Prilocaine was included in the preincubation at various concentrations to determine IC50 values. [2]

IC50 values for prilocaine were estimated from concentration-response curves at each temperature, as listed in Table I. [2]

NB2a mouse neuroblastoma cells were cultured in high glucose DMEM with glutamax-1, supplemented with 5% horse serum, 5% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 μg/ml), and gentamicin (25 μg/ml), in a humidified incubator at 37°C with 5% CO₂. For neurite outgrowth assay, cells were seeded onto 24-well plates at 15,000 cells/ml. After 24 hours, culture medium was replaced with serum-free medium plus 20 μl combinations of EGF and FGF containing prilocaine at concentrations of 1, 10, 25, or 100 μl. Cells were incubated for an additional 24 or 48 hours, then fixed with 4% formaldehyde in PBS for 10 minutes at room temperature, stained with Coomassie blue (0.6% Coomassie brilliant blue G in 10% acetic acid, 10% methanol, 80% PBS) for 3 minutes, washed with PBS, and photographed. Total neurite length per cell was measured using an image analyzer. [1]
- For cell viability assay, 6-well plates were seeded with 5×10⁴ cells in 1,500 μl medium, and 1, 10, 25, or 100 μl of prilocaine was added. After 24 hours, trypan blue (0.4%) was added to cell suspensions, and stained cells were counted in a Thoma cell counting chamber. The ratio of viable to dead cells was calculated. [1]
- For oxidative stress and apoptosis assessment, after serum-free medium plus EGF/FGF treatment, cells were fixed in 4% paraformaldehyde for 30 minutes, permeabilized with 0.1% Triton X-100 in PBS at 4°C for 15 minutes, then incubated with 3% H₂O₂ for 5 minutes to block endogenous peroxidase. Cells were incubated overnight at 4°C with anti-eNOS or anti-iNOS antibodies, then with biotinylated anti-rabbit IgG, then with horseradish peroxidase-conjugated streptavidin for 30 minutes each. Immunoreactivity was visualized using DAB and counterstained with Mayer's hematoxylin. Staining intensity was scored semi-quantitatively from 0 (no staining) to 5 (very strong). [1]
- Apoptosis was detected using the TUNEL method. Cells were treated with proteinase K for 10 minutes, then incubated with terminal deoxynucleotide transferase (TdT) at 37°C for 60 minutes. TdT was omitted for negative controls. TUNEL-positive cells were counted, and the percentage of apoptotic cells was calculated. [1]

Prilocaine produced concentration-dependent neurotoxicity in NB2a cells, with an inverse relationship between concentration and cell viability. At low concentrations, toxicity was manifested by neurite inhibition; at high concentrations, by apoptosis. [1]
- In comparison with other local anesthetics, prilocaine showed intermediate neurotoxicity: tetracaine (most toxic) > prilocaine > lidocaine > procaine (least toxic). [1]
- At 1 μl concentration, cell viability difference between procaine and prilocaine was significant (p < 0.05). At 10 μl, prilocaine caused significantly lower viability than procaine (p < 0.01) and higher toxicity than lidocaine (p < 0.05). [1]
- Prilocaine induced TUNEL-positive apoptotic cells, with significant difference compared to procaine (p < 0.01). Apoptosis was associated with increased i-NOS and e-NOS expression, indicating oxidative stress involvement. [1]

[1]. Neurotoxic effects of local anesthetics on the mouse neuroblastoma NB2a cell line. Biotech Histochem. 2015 Apr;90(3):216-22.

[2]. Effects of local anaesthetics on the activity of the Na,K-ATPase of canine renal medulla. Pharmacol Res. 2000 Jan;41(1):1-7.

Prilocaine hydrochloride is the hydrochloride form of prilocaine, a medium-acting amide-type local anesthetic with a chemical structure similar to lidocaine. Prilocaine hydrochloride binds to voltage-gated sodium ion channels on neuronal membranes, thereby preventing sodium ion permeability. This leads to neuronal membrane stabilization, inhibits depolarization, and ultimately reversibly blocks the generation and conduction of nerve impulses along nerve fibers, resulting in reversible loss of sensation. It is a local anesthetic with pharmacological effects similar to lidocaine. Currently, it is most commonly used for infiltration anesthesia in dentistry. See also: prilocaine (containing the active ingredient); epinephrine tartrate; prilocaine hydrochloride (ingredient).

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Prilocaine is an amide-type local anesthetic. The study found no relation between neurotoxicity and the ester/amide type, as both ester (tetracaine, procaine) and amide (lidocaine, prilocaine) types gave similar results. [1]
- The mechanism of local anesthetic-induced neurotoxicity remains unclear, but neurite inhibition can serve as a marker for moderate toxicity. Procaine was the least neurotoxic, and because it is short-acting, it may be preferred for pain prevention during short procedures. Prilocaine was not recommended as a less neurotoxic option in this study. [1]

C13H21CLN2O

256.77

256.134

1786-81-8

Prilocaine;721-50-6;Prilocaine-d7 hydrochloride;Prilocaine acetate

92163

Typically exists as solid at room temperature

85°C 4mm

168-170ºC

134.3ºC

2.05E-05mmHg at 25°C

3.587

3

2

5

17

218

0

Cl[H].O=C(C([H])(C([H])([H])[H])N([H])C([H])([H])C([H])([H])C([H])([H])[H])N([H])C1=C([H])C([H])=C([H])C([H])=C1C([H])([H])[H]

BJPJNTKRKALCPP-UHFFFAOYSA-N

InChI=1S/C13H20N2O.ClH/c1-4-9-14-11(3)13(16)15-12-8-6-5-7-10(12)2/h5-8,11,14H,4,9H2,1-3H3,(H,15,16)1H

N-(2-methylphenyl)-2-(propylamino)propanamidehydrochloride

Prilocaine HydrochloridePropitocaine hydrochloridePrilocaine HClXylonestCitanest OctapressinPrilocaine chlorideXylonest

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)