Voltage-gated sodium channels (inhibitor) - inferred from its classification as a local anesthetic, but not directly assayed in this study. [1]

Within 4 minutes in PC12 cells, butamben (500 μM) inhibits 90% of control barium currents [2]. Fast but not slow Na+ channel inactivation is increased by butamben (100 μM; 2–10 minutes) [3].

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Cytotoxicity (MTT assay): The cytotoxicity of various butamben gel formulations was assessed in BALB/c 3T3 mouse fibroblast cells. Cells were exposed to formulations containing different BTB concentrations (0.312 to 6.25 mM) for 2 hours. Cell viability was measured via the MTT assay. The half-maximal inhibitory concentration (IC50) values were calculated as 1.47 ± 0.22 mM for plain BTB gel, 2.63 ± 0.64 mM for BTB encapsulated in conventional liposomes (BTB_LUV gel), and 1.95 ± 0.42 mM for BTB encapsulated in elastic liposomes (BTB_LUV-EL gel). The IC50 values for the liposomal formulations were significantly higher than for the plain BTB gel (p<0.001 for BTB_LUV; p<0.01 for BTB_LUV-EL), indicating reduced cytotoxicity. Placebo gel formulations (without BTB) did not affect cell viability at the concentrations tested. [1]

In the mouse radiant heat tail flick test, butamben (0.5–50 mM; distal tail immersion for 2 minutes) exhibits dose-dependent analgesic efficacy in the form of a S [4].

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Topical Analgesia (Tail Flick Test): The analgesic effect of butamben gel formulations was evaluated in Swiss mice using the tail flick test. A 0.1 g sample of gel formulation (10% BTB) was applied topically to the base of the tail. The latency of tail withdrawal from a thermal stimulus (55 ± 1°C) was measured every 10 minutes until it returned to baseline. Both liposomal BTB gel formulations (BTB_LUV and BTB_LUV-EL) showed a significantly increased topical analgesic effect compared to the plain BTB gel (p<0.05). The duration of sensory block (recovery time) was 59 ± 8.9 min for plain BTB gel, 73.3 ± 20.4 min for BTB_LUV gel (24.2% increase), and 86.7 ± 22.3 min for BTB_LUV-EL gel (46.9% increase). The area under the efficacy curve (AUC_0-115) was also significantly larger for the liposomal formulations (4085.1 ± 1828 for BTB_LUV and 4166.3 ± 1176 for BTB_LUV-EL) compared to the plain BTB gel (1505.6 ± 582). Placebo gels induced no analgesia. [1]
Local Toxicity (Histopathology): The local toxicity of butamben gel formulations was assessed in Wistar rats. Gels (0.1 g) were applied topically to a shaved area (2 cm²) on the dorsum for 6 hours. After sacrifice, skin samples were excised, fixed, sectioned, and stained with H&E for histopathological analysis. No morphological changes in skin structures (epidermis, stratum corneum, hair follicles, sebaceous glands) or evidence of cell infiltration were observed for any of the tested formulations, including plain BTB gel, liposomal BTB gels, and placebo gels. [1]

Whole-Cell Patch-Clamp on DRG Neurons: Cultured neonatal rat dorsal root ganglion (DRG) neurons (1 day in culture, diameter 10-15 μm) were used. Experiments were performed at room temperature (19°C). Borosilicate glass patch pipettes (resistance ~3-4 MΩ) were filled with intracellular solutions designed to block K+ currents (e.g., containing Cs+ and TEA+). Cells were bathed in extracellular solutions that could be quickly changed via a perfusion system. Current-clamp and voltage-clamp protocols were applied using a pulse generator and an EPC-7 amplifier. Signals were filtered at 10 kHz, digitized, and stored for analysis. Leakage and capacity currents were subtracted digitally. [3]
Current-Clamp Protocol: The firing threshold was determined by applying 10-20 depolarizing current pulses (10 ms) with increasing amplitude at 1 Hz. Supramaximally evoked action potentials were recorded every 6 or 30 seconds to monitor drug effects. [3]
Voltage-Clamp Protocols:
Activation Protocol: A 40-ms prepulse to -140 mV to remove inactivation, followed by an 8-ms test pulse increasing from -100 to +100 mV in 10 mV steps (1 Hz).
Inactivation Protocol: A 40-ms prepulse varying from -140 to 0 mV in 5 mV steps to set the level of inactivation, followed by a test pulse to -10 mV. [3]
Data Analysis: Sodium currents were corrected for leakage and capacity currents. Peak sodium conductance (gpT) was calculated using the formula gpT = I_p,max / (E - E_Na). Steady-state inactivation curves (h∞) were fitted with a Boltzmann equation: I/Imax = 1/(1 + exp((E - E50)/k)). [3]

Topical Analgesia (Tail Flick Test) in Mice:** Male Swiss mice (35-45 g) were used. Baseline reaction times to a thermal stimulus (55 ± 1°C) were recorded the day before the experiment. On the test day, mice were divided into groups (n=7 per group) to receive different treatments: plain BTB gel, BTB_LUV gel, BTB_LUV-EL gel, base gel, LUV gel, and LUV-EL gel. A 0.1 g sample of the gel formulation was applied topically to the base of the tail. The tail was then exposed to the thermal stimulus every 10 minutes, and the latency to tail flick was recorded, with a cutoff time of 15 seconds to prevent injury. Testing continued until the latency time returned to the baseline value. The percentage of maximum possible effect (%MPE) was calculated. All experiments were performed by a blinded observer. [1]
* **Local Toxicity (Histopathology) in Rats:** Male Wistar rats (250-350 g) were divided into groups (n=6 per group) for treatment with the same formulations as in the mouse study. Animals were anesthetized with intraperitoneal urethane (1 g/kg) and alpha-chloralose (50 mg/kg). The dorsal area was shaved (2 cm²), and 0.1 g of the gel formulation was applied. After 6 hours, animals were sacrificed under deep anesthesia. The treated skin area was immediately excised, fixed in Bowin's solution for 24 hours, embedded in paraffin, and sectioned (5 μm thickness). Sections were stained with hematoxylin and eosin (H&E) and examined under a light microscope by a blinded assessor for structural changes and cell infiltration. [1]

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Topical Analgesia (Tail Flick Test) in Mice: Male Swiss mice (35-45 g) were used. Baseline reaction times to a thermal stimulus (55 ± 1°C) were recorded the day before the experiment. On the test day, mice were divided into groups (n=7 per group) to receive different treatments: plain BTB gel, BTB_LUV gel, BTB_LUV-EL gel, base gel, LUV gel, and LUV-EL gel. A 0.1 g sample of the gel formulation was applied topically to the base of the tail. The tail was then exposed to the thermal stimulus every 10 minutes, and the latency to tail flick was recorded, with a cutoff time of 15 seconds to prevent injury. Testing continued until the latency time returned to the baseline value. The percentage of maximum possible effect (%MPE) was calculated. All experiments were performed by a blinded observer. [1]
Local Toxicity (Histopathology) in Rats: Male Wistar rats (250-350 g) were divided into groups (n=6 per group) for treatment with the same formulations as in the mouse study. Animals were anesthetized with intraperitoneal urethane (1 g/kg) and alpha-chloralose (50 mg/kg). The dorsal area was shaved (2 cm²), and 0.1 g of the gel formulation was applied. After 6 hours, animals were sacrificed under deep anesthesia. The treated skin area was immediately excised, fixed in Bowin's solution for 24 hours, embedded in paraffin, and sectioned (5 μm thickness). Sections were stained with hematoxylin and eosin (H&E) and examined under a light microscope by a blinded assessor for structural changes and cell infiltration. [1]

Absorption, Distribution and Excretion
When butaben is administered epidurally in suspension form, its physical properties allow for slow release. Systemic absorption of butaben is reportedly very low with local administration, thus prolonging the duration of action. After metabolism by cholinesterase, the metabolites in plasma are primarily excreted in the urine. The pharmacokinetic properties of this drug have not been determined. Clearance is limited by blood flow and highly dependent on its protein binding state. Metabolism/Metabolites Butaben's metabolic pathway is similar to other local anesthetics, primarily through cholinesterase hydrolysis to produce inert metabolites. Biological Half-Life The effective half-life of unencapsulated butaben is 90 minutes. Attempts have been made to prepare D,L-lactate capsules to extend the half-life of butaben to 400 hours.

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It mentions that butamben has low water solubility (140 mg/L) and a pKa of 2.52. Previous work by the group (Cereda et al., 2013) established that encapsulation in liposomes, especially elastic ones, improves the skin permeation ability of BTB. The encapsulation rates of BTB in conventional and elastic liposomes were 71.7 ± 2.4% and 82.9 ± 1.6%, respectively. [1]

Protein Binding
Like all other local anesthetics, butaben is expected to bind highly to plasma proteins, primarily α-1-acid glycoprotein. Interactions These drugs (cholinesterase inhibitors, such as antimyasthenia gravis drugs; cyclophosphamide; demecalin; ethion; neurotoxic insecticides, possibly including large doses of topical malathion; isoflurane; thiotepa) may inhibit the metabolism of ester derivatives; patients receiving cholinesterase inhibitors may have an increased risk of toxicity due to absorption of large amounts of ester derivatives. /Local Anesthetics/
Metabolic products of PABA derivative local anesthetics may antagonize the antibacterial activity of sulfonamides, especially in cases of prolonged and large-volume absorption of anesthetics. /Local Anesthetics/
Non-human Toxicity Values Intraperitoneal LD50 in mice: 67 mg/kg

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In Vitro Cytotoxicity: The study directly assessed the in vitro cytotoxicity of butamben formulations. The IC50 values (see In Vitro section) demonstrate that encapsulation in liposomes significantly reduces the drug's cytotoxicity towards 3T3 fibroblasts. The elastic liposome formulation, despite showing better skin permeation in prior studies, exhibited higher cytotoxicity (lower IC50 of 1.95 mM) than the conventional liposome formulation (IC50 of 2.63 mM). [1]
In Vivo Local Toxicity: Histopathological analysis of rat skin after a 6-hour topical application of all butamben gel formulations revealed no morphological changes or signs of irritation, indicating good local tolerability. [1]

[1]. Liposomal butamben gel formulations: toxicity assays and topical anesthesia in an animal model. J Liposome Res. 2017 Mar;27(1):74-82.

[2]. The local anesthetic butamben inhibits total and L-type barium currents in PC12 cells. Anesth Analg. 2008 Jun;106(6):1778-83.

[3]. The local anesthetic n-butyl-p-aminobenzoate selectively affects inactivation of fast sodium currents in cultured rat sensory neurons. Anesthesiology. 1995 Jun;82(6):1463-73.

[4]. Analgesic synergy between topical morphine and butamben in mice. Anesth Analg. 2003 Oct;97(4):1103-7, table of contents.

Butyl p-aminobenzoate is a yellow powder, insoluble in water. (NTP, 1992)
Butylaminobenzoate is an amino acid ester formed by the condensation of the carboxyl group of 4-aminobenzoic acid and the hydroxyl group of 1-butanol. Its local anesthetic properties have been used for surface anesthesia of the skin and mucous membranes, as well as for relieving pain and itching caused by certain anorectal diseases. It is a local anesthetic. It is a benzoic acid ester, substituted aniline, amino acid ester, and primary amine compound. It is functionally related to 4-aminobenzoic acid and 1-butanol. It is the conjugate base of butylaminobenzoate (1+).
Butylaminobenzoate is a local anesthetic that exists in the form of butyl p-aminobenzoate. Its structure conforms to the standard molecular structure common in most local anesthetics, consisting of hydrophilic and hydrophobic domains linked by an intermediate ester bond. Due to its extremely low water solubility, butylaminobenzoate is considered to be only suitable for local anesthesia. The U.S. Food and Drug Administration (FDA) has withdrawn all injectable butylaminobenzoate products from the market, possibly due to the drug's poor solubility.

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Drug Indications
Butaben, due to its long-acting effect, has been used to treat chronic pain. It is also used as a topical anesthetic for the skin and mucous membranes, and to relieve pain and itching associated with anorectal diseases.Mechanism of Action
Butaben works by inhibiting voltage-gated calcium channels in dorsal root ganglion neurons. This alteration of channels is thought to lead to dysregulation of channel dynamics. Butaben has also been reported as a sodium channel inhibitor and a potassium current delay rectifier. All of butaben's effects occur in nerve root ganglion neurons, suggesting that its associated anesthetic effect may be caused by reducing the electrical excitability of neurons.
...Its action is to interfere with the initiation and conduction of nerve impulses. Current theory suggests that local anesthetics can prevent nerve membrane depolarization, thereby preventing impulse propagation. ...This is thought to be due to interference with the exchange of sodium and potassium ions across the membrane.Therapeutic Uses
Local Anesthetics
...Poor water solubility, therefore extremely slow absorption, non-toxic.
It can be applied directly to the surface of wounds and ulcers and remains on the surface for an extended period…thus providing sustained anesthetic effects. …The most important member of this series is…Butylaminobenzoate (USP).
Dosage (Veterinary): For topical use, as a spray or ointment (1-2%). …Parasitual administration in oil form can provide anesthetic effects for up to 1 to 2 days, and is occasionally used for deep perianal injections requiring prolonged restraint (or in combination with procaine and benzyl alcohol).
Local anesthetics are indicated for the relief of pain, itching, and inflammation associated with minor skin conditions, including: minor burns (including sunburn); insect bites (or stings); contact dermatitis (including dermatitis caused by poison ivy, poison oak, or poison sumac); minor wounds such as cuts and abrasions. /Included in the US product label; Local Anesthetic/
Pharmacodynamics
Studies have shown that when administered as an epidural suspension, butylphenamine selectively inhibits the transmission of dorsal root pain signals, with effects lasting for months. Butanben's effects are not associated with any significant loss of motor function, suggesting that it specifically targets the pain-sensing C fibers of the dorsal root. When applied topically, butanben produces an anesthetic effect by accumulating on the nerve cell membrane, causing the cell membrane to dilate and lose its depolarizing ability, thereby blocking the transmission of nerve impulses.

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Background and Clinical Use: Butamben is described as a long-acting experimental local anesthetic. When administered epidurally as a suspension, it produces long-lasting selective segmental analgesia for the treatment of chronic pain, including intractable cancer pain. [3]
Mechanism of Action Related to Analgesia: This study elucidates the mechanism by which butamben affects sensory neuron excitability. The primary target appears to be fast (TTX-sensitive) sodium channels. Butamben does not block the channels in their resting state but instead causes a hyperpolarizing shift in their voltage-dependent inactivation. This means that at normal resting membrane potentials, a larger fraction of fast sodium channels are inactivated and unavailable to open, thereby raising the threshold for action potential firing and reducing neuronal excitability. The slow sodium channels are relatively unaffected. This selective action on fast sodium channels, combined with the slow washout after prolonged exposure, may explain its long-lasting analgesic effect. The study suggests that the small neurons used (10-15 μm) likely correspond to pain-transmitting Aδ and C fibers. [3]

193.11

94-25-7

Butamben-d9

2482

White to off-white solid powder

1.1±0.1 g/cm3

325.7±0.0 °C at 760 mmHg

57-58 °C(lit.)

184.6±17.9 °C

0.0±0.7 mmHg at 25°C

1.540

3.01

1

3

5

14

174

0

O=C(C1C=CC(N)=CC=1)OCCCC

IUWVALYLNVXWKX-UHFFFAOYSA-N

InChI=1S/C11H15NO2/c1-2-3-8-14-11(13)9-4-6-10(12)7-5-9/h4-7H,2-3,8,12H2,1H3

butyl 4-aminobenzoate

PlanoformScuroformButambenButesinZyljectinNSC128464ButanylcaineButoformNSC 128464NSC-128464

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)

DMSO : ≥ 100 mg/mL (~517.49 mM)
H2O : ~0.1 mg/mL (~0.52 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (12.94 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 (12.94 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 25.0 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: ≥ 2.5 mg/mL (12.94 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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 (Please use freshly prepared in vivo formulations for optimal results.)