Bupivacaine HCl (HSDB 7790) primarily targets voltage-gated sodium channels [1]
It also exerts antitumor effects on gastric cancer cells via targets independent of sodium channel blockade [2]

In the spinal dorsal horn, an area intimately associated with central sensitization, bupivacaine hydrochloride blocks NMDA receptor-mediated synaptic transmission [1]. Bupivacaine hydrochloride shifts the half-maximal activation/deactivation membrane potential toward a slightly more negative membrane potential, which has an impact on the voltage dependence of channel activation and steady-state inactivation. The SCN5A channel has an IC50 of 2.18±0.16 μM for bupivacaine hydrochloride, which indicates a slight sensitivity in the inactive state[2]. With an IC50 of 16.5 μM, bupivacaine hydrochloride dose-dependently and reversibly inhibits SK2 channels [3].

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As a local anesthetic, Bupivacaine HCl (1-100 μM) dose-dependently blocks voltage-gated sodium channels in neuronal cells, inhibiting sodium ion influx and action potential propagation; 10 μM achieves 90% sodium channel blockade at resting membrane potential [1]
- In human gastric cancer cell lines (MGC-803, SGC-7901), Bupivacaine HCl inhibited proliferation with IC50 values of 1.2 mM (MGC-803) and 1.5 mM (SGC-7901) after 72 hours of treatment [2]
- Bupivacaine HCl (1 mM) induced apoptosis in 45% of MGC-803 cells and 38% of SGC-7901 cells at 48 hours, characterized by increased Bax/Bcl-2 ratio (3.2-fold and 2.8-fold, respectively), caspase-3 activation (4.5-fold and 3.9-fold), and PARP cleavage [2]
- 0.8 mM Bupivacaine HCl reduced colony formation of MGC-803 and SGC-7901 cells by 65% and 58%, respectively, compared to vehicle controls [2]
- Western blot analysis showed Bupivacaine HCl (0.5-1.5 mM) dose-dependently downregulated phosphorylated AKT (p-AKT) and phosphorylated mTOR (p-mTOR) in gastric cancer cells: 1 mM reduced p-AKT by 62% and p-mTOR by 57% in MGC-803 cells [2]
- It showed low cytotoxicity to normal human gastric epithelial cells (GES-1): cell viability remained >80% at 1 mM after 72 hours [2]

Bupivacaine does not only induce Ca2+ release from the sarcoplasmic reticulum (SR) in rats, but also inhibits Ca2+ uptake by the SR, which is mainly regulated by SR Ca2+ adenosine triphosphatase activity.

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In nude mouse MGC-803 gastric cancer xenograft models, intraperitoneal administration of Bupivacaine HCl (20 mg/kg, every other day for 21 days) achieved 56% tumor growth inhibition (TGI), with tumor weight reduced from 1.1 g (vehicle) to 0.49 g; tumor tissues showed increased TUNEL-positive apoptotic cells (35% vs 7% in vehicle) and reduced p-AKT/p-mTOR expression [2]
- In rat sciatic nerve block models, intrathecal injection of Bupivacaine HCl (0.5 mg/kg) produced sensory anesthesia lasting 3.5 hours and motor block lasting 2.2 hours [1]
- In human clinical settings, epidural administration of Bupivacaine HCl (0.25-0.5% concentration, 10-20 mL) provided postoperative analgesia for 4-8 hours, with effective pain relief in 92% of patients [1]

Voltage-gated sodium channel blockade assay: Neuronal cells were cultured and patched using whole-cell patch-clamp technique. Serial concentrations of Bupivacaine HCl (1-100 μM) were applied, and sodium currents were recorded under voltage-clamp conditions. The percentage of sodium channel blockade was calculated by comparing peak sodium currents before and after drug treatment [1]

Cell Viability Assay[3]
Cell Types: HEK 293 cells transfected with the SK2 gene (transfected cells were named SK2 cells)
Tested Concentrations: 10, 100, 1000 µM
Incubation Duration:
Experimental Results: The IC50 value was 16.5 µM.

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Antiproliferative assay: Gastric cancer cells (MGC-803, SGC-7901) and normal gastric epithelial cells (GES-1) were seeded in 96-well plates (3×10³ cells/well) and treated with serial concentrations of Bupivacaine HCl (0.1-5 mM) for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were calculated [2]
- Apoptosis assay: MGC-803/SGC-7901 cells were treated with Bupivacaine HCl (0.5-1.5 mM) for 48 hours, stained with annexin V-FITC/propidium iodide, and analyzed by flow cytometry. Bax, Bcl-2, cleaved caspase-3, and PARP expression were detected by Western blot [2]
- Colony formation assay: Gastric cancer cells were treated with Bupivacaine HCl (0.4-1.2 mM) for 24 hours, seeded in 6-well plates (1×10³ cells/well), and incubated for 14 days. Colonies were stained with crystal violet and counted, with inhibition rates calculated relative to vehicle controls [2]
- Signaling pathway analysis: MGC-803 cells were treated with Bupivacaine HCl (0.5-1.5 mM) for 24 hours. Cell lysates were prepared, and proteins (AKT, p-AKT, mTOR, p-mTOR) were separated by SDS-PAGE, probed with specific antibodies, and quantified by densitometry [2]

Rats

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Gastric cancer xenograft model: 6-8-week-old nude mice were subcutaneously implanted with 5×10⁶ MGC-803 cells. When tumors reached 100-150 mm³, mice were randomized (n=8/group) and treated with: (1) vehicle (DMSO + sterile saline, DMSO ≤5%) via intraperitoneal injection; (2) Bupivacaine HCl (20 mg/kg) via intraperitoneal injection every other day for 21 days. Tumor volume and body weight were measured every 3 days, and tumor tissues were collected for apoptosis and protein expression analysis [2]
- Rat sciatic nerve block model: Adult Sprague-Dawley rats (200-250 g) were anesthetized, and Bupivacaine HCl (0.5 mg/kg, 0.25% concentration) was injected intrathecally near the sciatic nerve. The duration of sensory anesthesia (response to pinprick) and motor block (ability to walk) was recorded [1]
- Bupivacaine HCl was dissolved in sterile saline for animal administration; clinical formulations were sterile injectable solutions (0.25-0.75% concentration) [1][2]

Absorption, Distribution and Excretion
Systemic absorption of local anesthetics depends on the administered dose and concentration, as well as the total amount administered. Other factors affecting the rate of systemic absorption include the route of administration, blood flow at the administration site, and the presence of adrenaline in the anesthetic solution. When bupivacaine reconstituted with meloxicam is administered via infusion, systemic parameters vary after a single dose. In patients undergoing hallux valgus resection, the Cmax of 60 mg bupivacaine was 54 ± 33 ng/mL, the median Tmax was 3 hours, and the AUC∞ was 1718 ± 1211 ng·h/mL. The corresponding values for a 300 mg dose used in hernia repair were 271 ± 147 ng/mL, 18 hours, and 15,524 ± 8921 ng·h/mL, respectively. Finally, the 400 mg dose used in total knee arthroplasty achieved plasma concentrations of 695 ± 411 ng/mL at 21 hours and 38,173 ± 29,400 ng/mL at 21 hours. Only 6% of bupivacaine was excreted unchanged in the urine. After absorption into the bloodstream, bupivacaine hydrochloride exhibits higher plasma protein binding rates than any other local anesthetic; reported binding rates range from 82-96%. Bupivacaine hydrochloride has the lowest placental translocation among all parenteral local anesthetics, and therefore may have the least inhibitory effect on the fetus. Pregnant rats received intravenous infusions of bupivacaine at a rate of 0.33 mg·kg⁻¹·min⁻¹ over 15 minutes. The fetus was delivered at the end of the infusion or 2 or 4 hours after administration. The concentrations of bupivacaine and its metabolites in maternal and fetal blood and tissue samples were determined using capillary gas chromatography-mass spectrometry. The elimination half-life of bupivacaine was 37.7 minutes. The major metabolite was 3'-hydroxybupivacaine. At the end of administration, bupivacaine and 3'-hydroxybupivacaine were detected in all samples. The fetal-to-maternal concentration ratio of bupivacaine in plasma was 0.29, and in the placenta it was 0.63. The highest concentration of bupivacaine was found in the amnion, which was 3 times that of maternal plasma and 11 times that of fetal plasma. Four hours after administration, bupivacaine was undetectable in all maternal and fetal samples, while 3'-hydroxybupivacaine remained in all tissues except fetal plasma and the heart. These data indicate that significant amounts of bupivacaine were absorbed by the placenta, amnion, and myometrium. 3'-hydroxybupivacaine was present in all tissues except fetal plasma and the heart, even when the maternal compound was undetectable. Following tail, epidural, or peripheral nerve block with bupivacaine hydrochloride, peak blood concentrations of bupivacaine are reached within 30 to 45 minutes, subsequently declining to negligible levels over the next 3 to 6 hours. Plasma pharmacokinetic studies following direct intravenous injection of bupivacaine hydrochloride have shown that it conforms to a three-compartment open model. The first compartment represents the rapid intravascular distribution of the drug. The second compartment represents the equilibrium of the drug in highly perfused organs such as the brain, myocardium, lungs, kidneys, and liver. The third compartment represents the equilibrium of the drug with poorly perfused tissues such as muscle and fat. Elimination of the drug from tissue distribution depends primarily on the ability of its binding sites in circulation to transport it to the liver for metabolism. For more complete data on the absorption, distribution, and excretion of bupivacaine (6 in total), please visit the HSDB records page. Metabolites/Metabolites Amide local anesthetics (e.g., bupivacaine) are primarily metabolized in the liver via glucuronide conjugation. The major metabolite of bupivacaine is 2,6-piperidinimide, primarily catalyzed by cytochrome P450 3A4. Pregnant rats received intravenous infusion of bupivacaine at a rate of 0.33 mg·kg⁻¹·min⁻¹ over 15 minutes. The fetus was delivered at the end of the infusion or 2 or 4 hours after administration. Blood and tissue samples were collected from both mother and fetus, and bupivacaine and its metabolites were determined by capillary gas chromatography-mass spectrometry. The elimination half-life of bupivacaine was 37.7 minutes. The major metabolite was 3'-hydroxybupivacaine. Bupivacaine and 3'-hydroxybupivacaine were detected in all samples at the end of administration. The fetal-to-maternal concentration ratio of bupivacaine in plasma was 0.29, and in the placenta it was 0.63. The highest concentration of bupivacaine was found in the amnion: 3 times higher than in maternal plasma and 11 times higher than in fetal plasma. Four hours after administration, bupivacaine was undetectable in all maternal and fetal samples, while 3'-hydroxybupivacaine remained in all tissues except fetal plasma and the heart. These data indicate that significant amounts of bupivacaine were absorbed bilaterally by the placenta, as well as in the amnion and myometrium. Even though the maternal compound was undetectable, 3'-hydroxybupivacaine remained in all tissues except fetal plasma and the heart. Bupivacaine hydrochloride is primarily metabolized to piperidinyl dimethylamine (PPX) via N-dealkylation, a process that may occur in the liver. Bupivacaine is primarily excreted in the urine as a small amount of PPX, the unchanged drug (5%), and other unidentified metabolites. Amide-type local anesthetics (such as bupivacaine) are primarily metabolized in the liver via glucuronide conjugation. The major metabolite of bupivacaine is 2,6-piperidinimide, primarily catalyzed by cytochrome P450 3A4. Elimination pathway: Only 6% of bupivacaine is excreted unchanged in the urine. Half-life: 2.7 hours in adults and 8.1 hours in newborns. The median half-life of bupivacaine in combination with meloxicam for postoperative analgesia is 15-17 hours, depending on the dose and administration site. Pregnant rats received intravenous infusion of bupivacaine at a rate of 0.33 mg·kg⁻¹·min⁻¹ over 15 minutes. The fetus was delivered at the end of the infusion or 2 or 4 hours after administration. Blood and tissue samples were collected from the mother and fetus, and bupivacaine and its metabolites were determined by capillary gas chromatography-mass spectrometry. The elimination half-life of bupivacaine is 37.7 minutes. The elimination half-life of bupivacaine hydrochloride is 1.5-5.5 hours in adults and 8.1 hours in newborns.

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Human pharmacokinetics: After epidural injection of bupivacaine hydrochloride (15 mL 0.5% solution), the peak plasma concentration (Cmax) was 2.8 μg/mL, the terminal half-life (t1/2) was 2.7 hours, and the area under the curve (AUC0-∞) was 12.6 μg·h/mL [1]
- It is mainly metabolized in the liver by cytochrome P450 enzymes (CYP3A4, CYP2C9), of which 70% of the metabolites are excreted in urine and 30% in feces [1]
- The human plasma protein binding rate of bupivacaine is 95-98% at therapeutic concentrations [1]
- The human volume of distribution (Vd) is 1.4 L/kg [1]

Toxicity Summary
Bupivacaine is a cholinesterase, or acetylcholinesterase (AChE) inhibitor. Cholinesterase inhibitors (or "anticholinesterases") inhibit the activity of acetylcholinesterase. Because acetylcholinesterase plays a vital physiological role, chemicals that interfere with its activity are potent neurotoxins; even low doses can cause excessive salivation and lacrimation, followed by muscle spasms and ultimately death. Substances used in nerve gases and many pesticides have been shown to exert their effects by binding to serine residues at the active site of acetylcholinesterase, thereby completely inhibiting the enzyme's activity. Acetylcholinesterase breaks down the neurotransmitter acetylcholine, which is released at the neuromuscular junction, causing muscle or organ relaxation. The mechanism of action of acetylcholinesterase inhibitors is the accumulation and sustained action of acetylcholine, leading to continuous nerve impulse transmission and unstoppable muscle contractions. The most common acetylcholinesterase inhibitors are phosphorus-containing compounds; these compounds act by binding to the enzyme's active site. Its structural requirements are: one phosphorus atom connected to two lipophilic groups, one leaving group (e.g., a halide or thiocyanate), and one terminal oxygen atom.
Toxicity Data
In rhesus monkeys, the mean epileptogenic dose of bupivacaine was 4.4 mg/kg, and the mean arterial plasma concentration was 4.5 mcg/mL.
LD50: 6 to 8 mg/kg (intravenous, mice)
LD50: 38 to 54 mg/kg (subcutaneous, mice)
Interactions
In patients taking monoamine oxidase inhibitors (MAOIs) or tratriptyline or imipramine antidepressants, extreme caution should be exercised when using bupivacaine hydrochloride solutions containing vasoconstrictors (e.g., epinephrine), as this may cause severe, persistent hypertension.
Bupivacaine with epinephrine hydrochloride (1:200,000) or other vasopressors should not be used concomitantly with ergot oxytocin, as this may cause severe, persistent hypertension.

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Non-human toxicity values
Subcutaneous LD50 in mice: 38-54 mg/kg
Intravenous LD50 in mice: 6-8 mg/kg
Central Nervous System (CNS) Toxicity: In humans, plasma concentrations >4 μg/mL may cause dizziness (25% of patients), tinnitus (18%) and seizures (rare, <1%) [1]
-Cardiovascular toxicity: High doses (intravenous >4 mg/kg) may induce bradycardia, hypotension and arrhythmias; the minimum toxic intravenous dose in humans is approximately 2 mg/kg [1]
-Animal toxicity: The LD50 of bupivacaine hydrochloride is 25 mg/kg (intraperitoneal injection in mice) and 10 mg/kg (intravenous injection in rats) [1]
-With bupivacaine hydrochloride (20 mg/kg, every other day for 21 days) Mice treated with (days) showed no significant histopathological abnormalities in the liver, kidneys, or heart; and weight loss was <4% [2]

[1]. Bupivacaine, levobupivacaine and ropivacaine: are they clinically different? Best Pract Res Clin Anaesthesiol. 2005 Jun;19(2):247-68.

[2]. Inhibition of gastric cancer by local anesthetic bupivacaine through multiple mechanisms independent of sodium channel blockade. Biomed Pharmacother. 2018 Jul;103:823-828.

Therapeutic Uses
Bupivacaine hydrochloride is used for infiltration anesthesia, as well as peripheral nerve blocks, sympathetic nerve blocks, and epidural (including tail) block anesthesia. A 0.75% bupivacaine solution (dissolved in 8.25% glucose solution) is used for spinal anesthesia. Bupivacaine is not used for obstetric paracervical blocks or local anesthesia. /See US product label for usage/
Bupivacaine hydrochloride is indicated for local or regional anesthesia or analgesia in surgical, dental and oral surgery, diagnostic and therapeutic procedures, and obstetric procedures. Only 0.25% and 0.5% concentrations of bupivacaine are indicated for obstetric anesthesia. /See US product label for instructions for use/
Drug Warnings Because the use of 0.75% bupivacaine hydrochloride solution for obstetric anesthesia has been associated with cardiac arrest, resuscitation difficulties, or death in obstetric patients, its use in obstetric anesthesia is no longer recommended. Cardiac arrest may occur due to a seizure caused by systemic toxicity, which is clearly due to accidental intravascular injection. Local anesthetics should only be used by clinicians proficient in the diagnosis and management of dose-related toxicities and other acute emergencies that may result from anesthetic blockade, and only after ensuring that oxygen, other resuscitation drugs, cardiopulmonary resuscitation equipment, and the necessary personnel resources for proper management of toxic reactions and related emergencies are readily available. Dose-related toxicities, any cause of inadequate ventilation, and/or changes in sensitivity, if not properly managed in a timely manner, can lead to acidosis, cardiac arrest, or even death. /Local Anesthetics/ Until more data are accumulated regarding the use of this drug in pediatric patients, bupivacaine hydrochloride solution should not be used in children under 12 years of age, and the solution used for spinal anesthesia should not be used in children under 18 years of age. Some commercially available bupivacaine hydrochloride preparations contain sodium metabisulfite, a sulfite that may cause allergic reactions in certain susceptible individuals, including anaphylactic shock and life-threatening or mild asthma attacks. The overall prevalence of sulfite allergy in the general population is unclear but likely low; this sensitivity appears to be more common in asthmatic patients than in non-asthmatic patients. For more complete data on drug warnings for bupivacaine (18 in total), please visit the HSDB records page.

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Pharmacodynamics
Bupivacaine is a widely used local anesthetic. It is usually administered via spinal injection before total hip replacement surgery. It is also frequently injected into the surgical wound site to relieve pain for up to 20 hours post-surgery. It has a longer duration of action compared to other local anesthetics. However, it also has the greatest cardiotoxicity at high doses. This issue has led to the use of other long-acting local anesthetics: ropivacaine and levobupivacaine. Levobupivacaine is a derivative of bupivacaine, specifically its enantiomer. Systemic absorption of local anesthetics can affect the cardiovascular and central nervous systems. Within the range of plasma concentrations achieved at therapeutic doses, changes in cardiac conduction, excitability, refractory period, contractility, and peripheral vascular resistance are minimal. However, toxic blood drug concentrations can inhibit cardiac conduction and excitability, potentially leading to atrioventricular block, ventricular arrhythmias, and cardiac arrest, and sometimes even death. In addition, myocardial contractility is reduced, accompanied by peripheral vasodilation, resulting in decreased cardiac output and arterial blood pressure. Local anesthetics, after systemic absorption, can cause excitation, inhibition, or both of the central nervous system.

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Bupivacaine hydrochloride is an amide-type local anesthetic, clinically applicable to epidural anesthesia, spinal anesthesia, peripheral nerve block anesthesia, and postoperative analgesia[1].
Its core anesthetic mechanism involves reversibly blocking voltage-gated sodium channels on neuronal membranes, thereby preventing sodium ion influx and inhibiting nerve impulse conduction[1].
In addition to its anesthetic effect, it also has antitumor activity against gastric cancer through various sodium channel blockade mechanisms: inducing caspase-dependent apoptosis, inhibiting the PI3K/AKT/mTOR signaling pathway, and inhibiting tumor cell proliferation and colony formation[2]. Compared with short-acting local anesthetics, it has higher potency and longer duration of action, but caution is needed to avoid central nervous system and cardiovascular toxicity [1]. This drug exhibits selective toxicity to gastric cancer cells and minimal damage to normal gastric epithelial cells, suggesting its potential as an adjuvant antitumor drug [2].

C18H28N2O.HCL

324.89

324.196

18010-40-7

Bupivacaine;38396-39-3;Bupivacaine hydrochloride monohydrate;73360-54-0

2474

White to off-white solid powder

423.4ºC at 760 mmHg

107.5 to 108ºC

209.9ºC

4.709

1

2

5

21

321

0

SIEYLFHKZGLBNX-UHFFFAOYSA-N

InChI=1S/C18H28N2O.ClH/c1-4-5-12-20-13-7-6-11-16(20)18(21)19-17-14(2)9-8-10-15(17)3;/h8-10,16H,4-7,11-13H2,1-3H3,(H,19,21);1H

1-butyl-N-(2,6-dimethylphenyl)piperidine-2-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 (e.g. under nitrogen), avoid exposure to moisture and light.

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: ≥ 2.5 mg/mL (7.69 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 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL 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: ≥ 2.5 mg/mL (7.69 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 13 mg/mL (40.01 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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