Voltage-gated sodium channels (VGSCs)

All cells die when exposed to 0.25 mM and 1 mM of veratridine for 24 hours [2]. A tetrodotoxin-sensitive reaction is elicited by veratridine (0.001-100 μM) [3].

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Veratridine (VTD) is a lipid-soluble neurotoxin derived from plants in the family Liliaceae. It has been broadly investigated for its action as a sodium channel agonist. However, the effects of veratridine on subtypes of sodium channels, especially Nav1.7, remain to be studied. Here, we investigated the effects of veratridine on human Nav1.7 ectopically expressed in HEK293A cells and recorded Nav1.7 currents from the cells using whole-cell patch clamp technique. We found that veratridine exerted a dose-dependent inhibitory effect on the peak current of Nav1.7, with the half-maximal inhibition concentration (IC50) of 18.39 µM. Meanwhile, veratridine also elicited tail current (linearly) and sustained current [half-maximal concentration (EC50): 9.53 µM], also in a dose-dependent manner. Veratridine (75 µM) shifted the half-maximal activation voltage of the Nav1.7 activation curve in the hyperpolarized direction, from -21.64 ± 0.75 mV to -28.14 ± 0.66 mV, and shifted the half-inactivation voltage of the steady-state inactivation curve from -59.39 ± 0.39 mV to -73.78 ± 0.5 mV. An increased frequency of stimulation decreased the peak and tail currents of Nav1.7 for each pulse along with pulse number, and increased the accumulated tail current at the end of train stimulation. These findings reveal the different modulatory effects of veratridine on the Nav1.7 peak current and tail current. [1]

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Veratridine (VTD) is a plant neurotoxin that acts by blocking the voltage-gated sodium channels (VGSC) of cell membranes. Symptoms of VTD intoxication include intense nausea, hypotension, arrhythmia, and loss of consciousness. The treatment for the intoxication is mainly focused on treating the symptoms, meaning there is no specific antidote against VTD. In this pursuit, we were interested in studying the molecular interactions of VTD with cyclodextrins (CDs). CDs are supramolecular macrocycles with the ability to form host–guest inclusion complexes (ICs) inside their hydrophobic cavity. Since VTD is a lipid-soluble alkaloid, we hypothesized that it could form stable inclusion complexes with different types of CDs, resulting in changes to its physicochemical properties. In this investigation, we studied the interaction of VTD with β-CD, γ-CD and sulfobutyl ether β-CD (SBCD) by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy. Docking and molecular dynamics studies confirmed the most stable configuration for the inclusion complexes. Finally, with an interest in understanding the effects of the VTD/CD molecular interactions, we performed cell-based assays (CBAs) on Neuro-2a cells. Our findings reveal that the use of different amounts of CDs has an antidote-like concentration-dependent effect on the cells, significantly increasing cell viability and thus opening opportunities for novel research on applications of CDs and VTD. [2]

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Nociceptors are a subpopulation of dorsal root ganglia (DRG) neurons that detect noxious stimuli and signal pain. Veratridine (VTD) is a voltage-gated sodium channel (VGSC) modifier that is used as an "agonist" in functional screens for VGSC blockers. However, there is very little information on VTD response profiles in DRG neurons and how they relate to neuronal subtypes. Here we characterised VTD-induced calcium responses in cultured mouse DRG neurons. Our data shows that the heterogeneity of VTD responses reflects distinct subpopulations of sensory neurons. About 70% of DRG neurons respond to 30-100 μM VTD. We classified VTD responses into four profiles based upon their response shape. VTD response profiles differed in their frequency of occurrence and correlated with neuronal size. Furthermore, VTD response profiles correlated with responses to the algesic markers capsaicin, AITC and α, β-methylene ATP. Since VTD response profiles integrate the action of several classes of ion channels and exchangers, they could act as functional "reporters" for the constellation of ion channels/exchangers expressed in each sensory neuron. Therefore our findings are relevant to studies and screens using VTD to activate DRG neurons [3].

The current study aims to determine the safety and efficacy of Veratridine (VTD) at doses sufficient to induce UBXN2A expression in a mouse model. A set of flow-cytometry experiments confirmed that VTD induces both early and late apoptosis in a dose-dependent manner. In vivo intraperitoneal (IP) administration of VTD at 0.1 mg/kg every other day (QOD) for 4 weeks effectively induced expression of UBXN2A in the small and large intestines of mice. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays on tissues collected from VTD-treated animals demonstrated VTD concentrations in the low pg/mg range. To address concerns regarding neuro- and cardiotoxicity, a comprehensive set of behavioral and cardiovascular assessments performed on C57BL/6NHsd mice revealed that VTD generates no detectable neurotoxicity or cardiotoxicity in animals receiving 0.1 mg/kg VTD QOD for 30 days. Finally, mouse xenograft experiments in athymic nude mice showed that VTD can suppress tumor growth. The main causes for the failure of experimental oncologic drug candidates are lack of sufficient safety and efficacy. The results achieved in this study support the potential utility of VTD as a safe and efficacious anti-cancer molecule [4].

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Veratridine (VTD) exhibits no outward neurotoxicity at 0.1 mg/kg [4]
The LD50 (lethal dose 50%) of Veratridine (VTD) is 1.35 and 4.9 mg/kg for mice injected intraperitoneally and subcutaneously, respectively. Considering this, we conducted a maximum tolerated dose (MTD) experiment and administered VTD IP at 0.1, 0.3, 0.5, and 1 mg/kg to C57BL/6NHsd mice. Animals were visually monitored for VTD-described neurotoxicity signs and symptoms based on previous reports. As reported by Otoom et al. we observed dose-dependent signs of neurotoxicity with increasing VTD dosages. At the lowest VTD dose of 0.1 mg/kg, no outward neurological symptoms were noted. At 0.3 mg/kg, a brief frozen state lasting less than one minute was observed. At 0.5 mg/kg, animals exhibited a frozen state that lasted several minutes and then resolved. At 1 mg/kg, the animals exhibited severe freezing and respiratory distress and thus were immediately euthanized. The absence of neurotoxic signs at 0.1 mg/kg is similar to results previously reported in rats by Meilman et al.

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Veratridine (VTD) induces expression of UBXN2A in vivo [4]
Based on the MTD results, we decided to examine whether 0.1 mg/kg of VTD can induce expression of UBXN2A in an animal model, which previously had only been shown at the cellular level. To test this, C57BL/6NHsd mice were administered either VTD 0.1 mg/kg IP QOD or ethanol (0.01%) control. After 30 days, small and large intestinal tissues were collected for RNA and protein studies (Fig. 2A). The results in Fig. 2 show that treatment with VTD significantly induces expression of UBXN2A in mouse large intestine tissues in both RNA (Fig. 2C, mean control = 1, mean VTD = 2.97, N = 6 per treatment, P < 0.05) and protein (Fig. 2D, E, P < 0.05, N = 4 per treatment) compared to controls that received ethanol vehicle. The qRT-PCR showed that VTD increases the RNA level of UBXN2A in the small intestine but failed to attain statistical significance (Fig. 2B, mean control = 1, mean VTD = 11.77, N = 6 per treatment, P = 0.0840). Interestingly, qRT-PCR revealed that the large intestine section, which includes the descending section of the mouse colon, has a more uniform response to VTD. Consequently, western blot (WB) experiments confirmed significant translation of UBXN2A RNA to protein in response to VTD (Fig. 2E and Table 2, Supplemental Materials). We observed differences in response to VTD treatment among mice which could be due to variability in drug delivery/metabolism per animal. Additionally, the morphological heterogeneity of colon tissues and non-linear nature of WB signals across samples could be two additional reasons for these differences. We used GAPDH to normalize the UBXN2A signal to show the level of UBXN2A per mouse. The results clearly show heterogeneity in the distal colon in individual mice. Further studies and alternative techniques can determine reasons behind these differences. Testing the low dose of VTD in an animal model confirmed that 0.1 mg/kg VTD can effectively elevate the level of UBXN2A in mouse colon tissues.

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Tissue bioanalysis shows no major accumulation of Veratridine (VTD) in an animal model [4]
Plasma and tissue concentrations of VTD were determined by LC–MS/MS at 1, 1.5, 2, 4, 8, 12, 24, 48, and 72 h. Plasma concentrations were negligible (<0.20 ng/ml) for all tested samples, suggesting a minimal systemic presence of VTD upon low-dose administration in mice. Tissue concentrations overall were low, with only pg/mg concentrations in all studied tissues. VTD concentrations in brain tissue peaked at 8 h but were rapidly eliminated (Fig. 3A). Heart concentrations peaked immediately and were eliminated by 12 h (Fig. 3B), and all other samples showed drug elimination by 72 h (Fig. 3C–E). The low, if not undetectable, VTD concentrations at 48 h suggest that there is a negligible risk for drug accumulation with chronic dosing in all tissues except the lung. The lung is known to be naturally permeable to all small-molecule drugs. Further studies will be conducted on lungs in the future. The clearance of VTD in this current study matches in vitro metabolic assays in rat liver microsomes

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Acute and repeated exposure to Veratridine (VTD) does not impact behavior in mice [4]
The timeline for the behavioral cohort is shown in Fig. 4A. No significant differences between Veratridine (VTD) and the control group were found in any behavioral measure (Table 1, Supplemental Materials). Locomotor testing revealed no effect of VTD within distance traveled (Fig. 4B) or velocity (Fig. 4C). Significant interactions were found between sex and time point in both distance traveled (F[3,84] = 6.429; P = 0.001) and velocity (F[3,84] = 6.403; P = 0.001), with females exhibiting greater exploratory behavior compared to males. This effect was expected, as females traditionally exhibit increased locomotor exploration compared to males in all forms of locomotor-based testing. Post hoc analysis revealed decreased distance traveled and decreased velocity between baseline and the other time points in all groups as well as at the 4-week time point in females. No effect of VTD was found on measures of motor coordination (Fig. 4D), strength (Fig. 4E), or nociception (Fig. 4F). A significant effect of time point was found by rotarod testing (F(1.609,45.055) = 45.378; P < 0.001), with post hoc analysis revealing training improvement from baseline to the other 3 time points. Training effects were also seen with the grid hang test (F[3,84] = 6.046; P = 0.004), with mice jumping from the grid at the 4-week time point in all groups; and with Von Frey testing (F[3,84] = 11.619; P < 0.001), with all mice habituating to the stimuli at both the 2-week and 4-week time points compared to baseline. No significant differences were found in any measure in novel object recognition testing (Fig. 4G).

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Veratridine (VTD) has no impact on cardiac function and exhibits a mild blood pressure-lowering effect [4]
The timeline for the cardiovascular assessments is represented in Fig. 5A. Ejection fraction (EF) from ultrasound indicated no difference at any time point between VTD and control animals (Fig. 5B). Furthermore, no difference at any time point was detected for cardiac output (CO, Fig. 5C), heart rate (HR, Fig. 5D), or stroke volume (SV, Fig. 5E). After 4 weeks of drug treatment, there was no significant difference in any of the echo parameters by t-test (Fig. 5F–I) and no effect of sex by two-way ANOVA on HR or EF (data not shown). A sex difference was noted in CO (F[1,12] = 9.906; P = 0.008) and SV (F[1,12] = 11.95; P = 0.005) by two-way ANOVA, but there was no effect of treatment. Sex difference in CO and SV is common in C57BL/6NHsd mice due to the size difference between the sexes.

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Veratridine (VTD) reduces tumor size [4]
Figure 7 presents data from the in vivo mouse xenograft model. Foxn1nu mice were subcutaneously implanted with iRFP-tagged HCT-116 colorectal cancer cells (Table 3, Supplemental Materials) and subsequently treated with 0.1 mg/kg VTD IP QOD or control beginning one day after tumor implantation (Fig. 7A). 3D ultrasound to calculate precise tumor volume followed by LI-COR near-infrared imaging to visualize the effects of VTD on primary tumor growth was conducted weekly over 5 weeks (Fig. 7B–E and Supplemental Fig. S1). After 5 weeks, VTD treatment resulted in a significant reduction of iRFP signal, with a mean total LI-COR signal in the VTD group of 1.68E7 a.u. and in the control group of 4.37E7 a.u., P = 0.035 (Fig. 7F). Tumor volume was also significantly reduced, with mean total tumor volume in the VTD group of 409.3 mm3 and in the control group of 1117 mm3, P = 0.028 (Fig. 7G). As previously shown at the cellular level, the presence of VTD led to the induction of apoptotic and necrotic tumors. The TUNEL staining of extracted xenograft tumors indicates that VTD induces cell death in tumor tissues with larger regions of apoptotic/necrotic tissues in mice treated with VTD (Fig. 7H, I). A set of WB experiments (Supplemental Fig. S2) confirmed VTD treatment increases protein level of UBXN2A while decreases the level of mortalin indicating the negative regulatory role of UBXN2A on mortalin. These data show that 0.1 mg/kg VTD QOD can effectively function as an anti-growth agent.

Isothermal Titration Calorimetry [1]
ITC was performed using a Microcal VP-ITC isothermal titration calorimeter at 298.2 K and atmospheric pressure. The instrument was calibrated electronically. The data were acquired with a computer software provided by Calorimetry Sciences Corp and analyzed using the one-site model. VTD/CD binding experiments were performed by injecting 10 µL aliquots with 240 s of separation of a CD solution (4 mM) into the sample cell containing VTD solution (200 μM). All experiments were performed with constant stirring (200 rpm) driven by a stepping motor coupled to the isothermal titration calorimeter. A 20 mM pH 6 tris buffer was used to prepare the solutions and all the solutions were degassed before the titration experiment. The CD concentrations for the experiment were chosen in order to work below the critical aggregation concentration (cac) of each CD, to ensure that the measured enthalpy change represents the complex formation without contributions from a simultaneous dissolution of the CD aggregates. In control experiments, 10 µL aliquots of a CD solution (4 mM) were injected into the sample cell containing tris buffer without the VTD toxin.

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NMR Spectroscopy [1]
The 1H-NMR and 2D 1H-1H ROESY spectra were recorded in D2O containing DCl 0.1% (v/v) at 400 MHz in a Varian NMR System 400 at 298 K using a 1:1 VTD:CD molar ratio for each cyclodextrin (β-CD, γ-CD or SBCD). All signals were referenced to internal HDO (4.79 ppm). The ROESY spectra were acquired with a mixing time of 400 ms and a relaxation delay of 1.8 s. Proton resonances of VTD, the pure CDs, and the inclusion complexes were assigned with the aid of standard COSY and HSQC experiments on the same solutions.

Cytotoxicity assay[2]
Cell Types: Neuro-2a cells
Tested Concentrations: 0.25 mM and 1 mM
Incubation Duration: 24 hrs (hours)
Experimental Results: Resulted in approximately 100% cell death (0% cell viability).

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Cell viability assay [3]
Cell Types: Cultured Mouse DRG Neuron
Tested Concentrations: 0.001, 0.1, 1, 10, 30 and 100 μM
Incubation Duration:
Experimental Results: The number of responding neurons increased in a concentration-dependent manner starting from a threshold of 1 μM .

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Neuro-2a Cell Viability Experiments [1]
Neuro-2a cells (ATCC, CCL131) were maintained in 10% fetal bovine serum (FBS) RPMI medium at 310 K in a 5% CO2 humidified atmosphere. For the experiments, cells were cultured in a 96-well microplate in 5% FBS RPMI medium at an approximate density of 34,000 cells per well for 24 h. A stock solution of VTD (1 mM) was prepared in MilliQ® water and adjusted to pH 2 for solubilization. Stock solutions of CDs (50 mM β-CD, 200 mM γ-CD, and 200 mM SBCD) were prepared in PBS. Prior to exposure to VTD and CDs, one half of the microplate was treated with 0.4 mM ouabain. Then, 10 μL of VTD and 10 μL of CDs at different concentrations were added into the wells both with and without the OB treatment (as a control to evaluate CDs toxicity) and incubated for 24 h. Each well had a final pH value of 6. Cell viability was assessed in triplicate experiments using the colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] MTT assay. Absorbance values were read at 570 nm using an automated multi-well scanning spectrophotometer. The cell viability values were normalized with respect to the viability of the control without OB treatment.

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Electrophysiology [1]
Whole-cell patch-clamp recordings were performed using an EPC/10 amplifier and Pulse Software at room temperature as previously described with a slight modification. Cells were bathed in an extracellular solution composed of the following (in mM): 150 NaCl, 5 KCl, 10 HEPES, 2.5 CaCl2, and 1 glucose (pH adjusted to 7.4 with NaOH). Patch pipettes were fabricated using a P-97 puller to achieve resistances of 2–3 MΩ when filled with the intracellular solution, which was composed of the following (in mM): 107 CsF, 10 NaCl, 2 MgCl2, 1 CaCl2, 10 HEPES, 10 EGTA, and 10 TEACl (pH adjusted to 7.2 with CsOH). Currents were filtered at 2.9 kHz and digitized at 10 kHz. Serious resistance was compensated by 70–80% during the whole-cell recording. TTX was stocked as 1 mM in water and diluted to 0.5 µM with the extracellular solution when used, and Veratridine (VTD) was stocked as 75 mM in ethanol and diluted to 1–75 µM with extracellular solution when used.

Animals and Veratridine (VTD) treatment [4]
\nFive different in vivo assessments were performed: maximum tolerated dose (MTD), behavioral, cardiovascular, blood/tissue concentration, and xenograft. C57BL/6NHsd mice were used for the MTD, behavioral, cardiovascular, and blood/tissue cohorts, and Foxn1nu athymic nude mice were used for the xenograft cohort. For each type of assessment, separate cohorts of mice were utilized; however, when possible and appropriate, animal data from cohorts were combined to reduce animal numbers needed. Animal numbers utilized for each set of experiments are included with their associated figure legends.\n

\nAll mice used in this study were housed with 3–4 animals per cage, kept at 22 °C on a standard light cycle (lights on from 10:00 to 22:00 h), and provided free access to water and standard rodent chow. All animal treatments and group assignments were randomized. Institutionally supported core facilities performed comprehensive assessments of the impact of Veratridine (VTD) on live animals. All data were collected, measured, and statistically analyzed by individuals within each facility who were blinded to animal treatment. Details about statistical methods used for each of the behavioral, cardiovascular, xenograft, and laboratory assessments can be found in Supplemental Materials.\n

\nIn the behavioral, cardiovascular, and xenograft cohorts, drug concentration was given to match the dose required to induce UBXN2A expression from previous experiments and the timeline/frequency of typical cancer treatment paradigms. The dose of Veratridine (VTD) was given based on mean weekly body weight (BW) by sex, and vehicle control was matched by volume.\n

\nIn the cardiovascular and behavioral cohorts, the mean BW of males was 26.7 ± 4.5 g and of females was 20.9 ± 2.3 g at the 4-week endpoint. In the xenograft cohort at the 5-week endpoint, the mean BW of males and females was 31.3 ± 2.6 and 26.2 ± 1.9 g, respectively. All animals were weighed at weekly intervals and monitored carefully for health. In the cardiovascular and behavioral cohorts, none of the mice exhibited weight loss at any weighing interval, and all were alive and healthy at the ~4-week endpoint. Some of the xenograft animals exhibited minor weight loss once tumors became large during the 5th week. However, all animals were active and hydrated and remained in the study until endpoint.\n

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\n\n\nMaximum tolerated dose (MTD) cohort [4]
\nIn the MTD cohort, we conducted a MTD experiment and administered Veratridine (VTD) IP at 0.1, 0.3, 0.5, and 1 mg/kg to C57BL/6NHsd mice. Animals were visually monitored for any signs or symptoms of neurotoxicity.\n

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\n\nBlood/tissue cohort [4]
\nIn the blood/tissue cohort, Veratridine (VTD) at 0.1 mg/kg was given by the IP route. One male and one female C57BL/6NHsd mouse aged 12–16 weeks was sacrificed at each timepoint for later bioanalysis. The timepoints were 1, 1.5, 2, 4, 8, 12, 24, 48, and 72 h after IP injection of 0.1 mg/kg Veratridine (VTD). Animals were anesthetized with isoflurane to effect and administered 10 µl of 1000IU heparin via retro-orbital intravenous injection just before euthanasia to enable the collection of unclotted blood by cardiac stick. Blood was collected and spun at 1000×g for 3 min. Plasma and tissues were collected and frozen for later bioanalysis. Tissues collected included brain, heart, kidney, spleen, and lung. Details about the bioanalyses performed can be found in Supplemental Materials.\n

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\n\nBehavioral and cardiovascular cohorts [4]
\nMice in the behavioral and cardiovascular cohorts received Veratridine (VTD) 0.1 mg/kg IP QOD for a total of 30 days. For the cardiovascular and behavioral cohorts, both male and female C57BL/6NHsd mice at 8–12 weeks of age were randomized into Veratridine (VTD) and control groups. For the behavioral cohort, N = 16 mice were randomized to each of the treatment groups, with equal numbers of mice for each sex. Behavioral assessments encompassed a comprehensive battery of tests to evaluate motor coordination and balance, limb strength, sensory and pain threshold, and working memory in mice during Veratridine (VTD) treatment. For the cardiovascular cohort, N = 8 mice were randomized to each of the Veratridine (VTD) and control groups, with equal numbers of each sex. Cardiovascular assessments involved weekly evaluation of cardiac function using echocardiogram followed by endpoint evaluation of arterial and intracardiac hemodynamics. A timeline of the behavioral and cardiovascular assessments can be found with their respective results figures. Detailed descriptions of the evaluations performed can be found in Supplemental Materials.\n

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\n\nXenograft cohort [4]
\nFor the xenograft cohort, N = 12 male and female athymic nude-Foxn1nu mice at 7–8 weeks of age were randomized into each of the Veratridine (VTD) and control groups. Mice received bilateral injections of 1 × 106 iRFP-tagged HCT-116 colorectal cancer cells suspended in 200 μl Hanks’ buffer free-FBS in the subcutaneous space over each hindquarter to induce tumors. Mice were treated with Veratridine (VTD) 0.1 mg/kg IP QOD for a total of 37 days beginning the day after tumor induction. The formation and progression of tumors were monitored weekly with near-infrared fluorescent imaging using a LI-COR Classic Imager with MousePOD accessory and 3D ultrasound volume reconstructions using high-frequency ultrasound weekly beginning at 2 weeks. Tissues were collected at endpoint for downstream immunohistochemistry and biological experiments. A detailed description of the performed methods including biological and histological techniques can be found in Supplemental Materials.

Absorption, Distribution and Excretion
This article provides data on the lipid solubility of the drug in the human body, its binding to human serum albumin, and its urinary excretion after oral administration. The structure-activity relationship of the drug's lipid solubility and pharmacological properties is discussed.

Interactions
This study investigated the effects of Veratridine on the resting membrane potential of intact crayfish (Procambarus clarkii) axons in the presence of different extracellular alkaline earth metal cations using intracellular microelectrodes. At extracellular pH values of 7, 6, and 5, the epigenetic binding order was: CA2+ > ≥ SR2+ > MG2+ > BA2+.

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6280 Rat Intraperitoneal LD50 3500 ug/kg Lung, pleural or respiratory: cyanosis; Lung, pleural or respiratory: respiratory depression; Gastrointestinal: changes in salivary gland structure or function, Journal of Pharmacology and Experimental Therapeutics, 78(238), 1943
6280 Mouse Intraperitoneal LD50 1350 ug/kg, Journal of the Society for Experimental Biology and Medicine, 76(847), 1951 [PMID:14844368]
6280 Mouse Subcutaneous LD50 6300 ug/kg Behavioral: seizures or effects on epileptic threshold; Behavioral: ataxia; Lung, pleural or respiratory: cyanosis, Journal of Pharmacology and Experimental Therapeutics, 113(89), 1955 [PMID:13234031]
6280 Canine LD50 Intravenous injection 19 mg/kg Cardiac: Arrhythmias (including conduction changes), Journal of Pharmacology and Experimental Therapeutics, 120(412), 1957 [PMID:13476366]

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Non-human toxicity excerpt
Eleven alkaloids extracted from Veratrum species have been compared to investigate their effects on membrane potential and conductance of giant axons in squid and crayfish. They can be divided into three groups. 1) Veratridine, civaridine, and proVeratridine A and B can cause membrane depolarization. The potency decreases in this order, and the concentrations of Veratridine and civaridine required to reach 50% maximum depolarization are estimated to be 3.3 × 10⁻⁵ M and 3.7 × 10⁻³ M, respectively. The depolarization induced by Veratridine is mainly due to the selective increase of sodium ion permeability of the cell membrane at rest and can be antagonized by tetrodotoxin. All of these drugs can effectively enhance and prolong the negative (depolarized) postpotential. 2) Veratridine, isorhodiola, muldamine (5-Veratridine-3β,11α-diol-11-acetate), and 5-Veratridine-3α-11α-diol (1×10⁻⁴) can block action potentials with almost no depolarization. 5-Veratridine-3β,11α-diol can block the increase in sodium and potassium ion conductance. 3) Cyclopamine, gervin, Rubigervin, and Veratridine have no effect on resting potential or action potential. The possible structure-activity relationship of these effects is discussed in this paper.
Exogenous application of 1×10⁻⁴ molar concentration of Veratridine to squid axons can induce membrane depolarization. During the initial depolarization process, the repetitive after-discharge induced by a single stimulus gradually disappears as the membrane further depolarizes and the action potential is blocked. Depolarization is mainly due to the increase in resting sodium ion permeability of the membrane. Veratridine can cause depolarization of excitatory cells and significantly increase the levels of adenosine 3',5'-monophosphate (cAMP) and guanosine 3',5'-monophosphate (cGMP) in mouse cerebral cortex culture sections. Symptoms of Veratridine and related alkaloid poisoning include salivation, diarrhea, vomiting (even in cattle), diuresis, post-excitation collapse, weak and irregular pulse, deep and slow breathing, ultimately leading to death from convulsions or paralysis. The lethal dose of fresh rhizomes for horses is 1 g/kg, and for cattle it is 2 g/kg.

[1]. Veratridine modifies the gating of human voltage-gated sodium channel Nav1.7. Acta Pharmacol Sin. 2018 Nov;39(11):1716-1724.

[2]. Supramolecular Complexes of Plant Neurotoxin Veratridine with Cyclodextrins and Their Antidote-like Effect on Neuro-2a Cell Viability. Pharmaceutics. 2022 Mar 9;14(3):598.

[3]. Veratridine produces distinct calcium response profiles in mouse Dorsal Root Ganglia neurons. Sci Rep. 2017 Mar 24;7:45221.

[4]. Pre-clinical safety and therapeutic efficacy of a plant-based alkaloid in a human colon cancer xenograft model. Cell Death Discov. 2022 Mar 28;8(1):135.

Veratridine is a steroidal compound with sodium channel regulating activity, its function being related to a benzoate compound. Benzoate compounds are found in plants of the genera Veratrum and Smilax. It activates sodium channels, extending their opening time beyond normal limits. Mechanism of Action: The effects of Veratridine on excitatory tissues are predictable, based on its ability to enhance sodium ion permeability. Its effects include, but are not limited to, the release of neurotransmitters, hormones, and drugs absorbed by nerve endings. Therapeutic Uses: It has been tested in patients with myasthenia gravis to enhance muscle responses to specific motor neuron stimuli.
Experimental use to alter sodium channel dynamics/PRC: in excitable membranes/

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In summary, our study shows that Veratridine (VTD) exhibits dose-dependent and use-dependent inhibition of the peak current of Nav1.7 and shifts the activation and steady-state inactivation curves of Nav1.7 toward hyperpolarization, while enhancing the sustained current and tail current, which may contribute to the associated Na+ influx. Our study reveals a novel mechanism of Veratridine-modified Nav1.7 gating. [1] We successfully demonstrated the formation of inclusion complexes between the alkaloid neurotoxin Veratridine (VTD) and natural β-cyclodextrin (β-CD), γ-cyclodextrin (γ-CD), and the anionic β-CD derivative SBCD. The equilibrium constants of β-CD, γ-CD, and SBCD were estimated to be 1500 M⁻¹, 7200 M⁻¹, and 8200 M⁻¹, respectively, with γ-CD and anionic SBCD being the most stable host molecules. ¹H-NMR and ¹H-¹H ROESY experiments confirmed that VTD was encapsulated in the cavities of each cyclodextrin, and the most stable orientation of VTD within the cyclodextrin was elucidated by molecular docking and molecular dynamics simulations. In vitro studies showed that the three cyclodextrins studied had similar antidote effects against VTD toxicity, and were able to protect the viability of Neuro-2a cells to varying degrees, depending on the type of cyclodextrin and the concentrations of cyclodextrin and VTD. To our knowledge, this is the first study on the interaction between VTD neurotoxin and cyclodextrin, and opens the door to further research on the effects of cyclodextrin on other lipid toxins. [2] In summary, this study showed that Veratridine (VTD) induces heterogeneous calcium responses in TTX-sensitive sensory neurons. The VTD response profile reflects different subpopulations of sensory neurons. These subpopulations overlap with, but are not identical to, those identified by classical functional nociceptive markers (Fig. 7). The OS and RD lineages were particularly enriched in nociceptors (neurons sensitive to at least one of the three agonists), while the SD lineage was enriched in non-nociceptors (neurons insensitive to none of the three agonists). Our results characterize in detail the effects of VTD on different subsets of DRG neurons. Our work is relevant to studies and screenings using VTD to activate DRG neurons. [3]

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A high-throughput drug screening showed that the natural plant alkaloid Veratridine (VTD) induces the expression of the anticancer protein UBXN2A in colon cancer cells. UBXN2A inhibits the expression of the heat shock protein mortalin, which plays a dominant role in cancer development, including epithelial-mesenchymal transition (EMT), cancer cell stemness, drug resistance, and apoptosis. VTD-dependent UBXN2A expression leads to mortalin inactivation in colon cancer cells, making VTD a potential targeted therapy for malignant tumors with high mortalin expression. VTD has been used clinically to treat hypertension in the past few decades. However, with the discovery of new antihypertensive drugs and concerns about potential neurotoxicity and cardiotoxicity, VTD has been discontinued for this purpose. [4]

C36H51NO11

673.79024

673.346

C, 64.17; H, 7.63; N, 2.08; O, 26.12

71-62-5

6280

White to off-white solid powder

1.45 g/cm3

814.5ºC at 760 mmHg

180ºC

446.4ºC

1.663

1.293

6

12

5

48

1340

14

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FVECELJHCSPHKY-YFUMOZOISA-N

InChI=1S/C36H51NO11/c1-19-6-11-26-31(3,40)35(43)25(17-37(26)16-19)33(42)18-34-24(32(33,41)15-27(35)38)10-9-23-30(34,2)13-12-28(36(23,44)48-34)47-29(39)20-7-8-21(45-4)22(14-20)46-5/h7-8,14,19,23-28,38,40-44H,6,9-13,15-18H2,1-5H3/t19-,23-,24-,25-,26-,27-,28-,30-,31+,32+,33+,34+,35-,36-/m0/s1

[(1R,2S,6S,9S,10R,11S,12S,14R,15S,18S,19S,22S,23S,25R)-1,10,11,12,14,23-hexahydroxy-6,10,19-trimethyl-24-oxa-4-azaheptacyclo[12.12.0.02,11.04,9.015,25.018,23.019,25]hexacosan-22-yl] 3,4-dimethoxybenzoate

NSC-7524; veratridine; 71-62-5; veratridin; 3-Veratroylveracevine; Veratrine (amorphous); Veratrine (amorphous) (VAN); CHEBI:28051; HSDB 4078; NSC7524; NSC 7524; Veratridine

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 (~148.41 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.71 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 (3.71 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 (3.71 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.)