Toxicity Summary
Identification and Uses: Poison dart frog toxin is a neurotoxic steroidal alkaloid, initially isolated from the Colombian poison dart frog, and later found in some passerine birds in New Guinea. It is currently believed that neither of these vertebrates synthesizes the toxin de novo, but rather obtains it from their diet. Human Studies: Poison dart frog toxin is a potent regulator of voltage-gated sodium channels, causing irreversible depolarization of nerves and muscles, atrial fibrillation, arrhythmias, and ultimately heart failure. Since its discovery, field researchers have also reported numbness after skin contact with the toxin. Animal Experiments: When P. terribilis is kept in captivity, the levels of poison dart frog toxin in its body tend to decrease, but even after up to six years of captivity, these frogs remain at least five times more toxic than other Phyllobates species used by local residents to make poison darts. Poison dart frog toxin was not detected in F1 generation frogs kept in captivity to maturity. Nerve and muscle tissues extracted from wild-caught frogs and non-toxic F1 generation frogs were insensitive to poison dart toxin. The regulatory sites controlling sodium channel activation and permeability appeared to be only slightly altered to prevent interaction with poison dart toxin, but remained sensitive to other sodium channel activators (veratrine, gray toxin), which frogs are not exposed to in nature.

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Interactions
Previously, two different types of local anesthetics (LAs) have been found to block poison dart toxin (BTX)-modified sodium channels: type I LAs, such as cocaine and bupivacaine, preferentially interact with open channels; while type II LAs, such as benzocaine and tricaine, interact with inactivated channels. In this paper, we describe our study of a third LA—tetracaine. Tetracaine is a dual blocker that binds strongly to closed channels but also binds to open channels to some extent during membrane depolarization. The enhanced inactivation effect of tetracaine on BTX-modified Na+ channels was determined by steady-state inactivation assays and dose-response curves. Steady-state inactivation reached its maximum at -70 mV, with an estimated half-maximal inhibitory concentration (IC50) of 5.2 μM and a Hill coefficient of 0.98, indicating that one tetracaine molecule binds to one inactivated channel. Tetracaine also effectively interacted with Na+ channels upon membrane depolarization; at +50 mV, the IC50 was estimated at 39.5 μM and the Hill coefficient was 0.94. Unexpectedly, charged tetracaine was the dominant active form blocking the inactivated channel. Furthermore, extracellular Na+ ions appeared to antagonize the blocking effect of tetracaine on the inactivated channel. Consistent with these results, N-butyltetracaine quaternary ammonium salt (a permanently charged tetracaine derivative) remains a potent inactivation enhancer. Another tetracaine derivative, 2-(dimethylamino)ethylbenzoate lacking the 4-butylamino functional group on the benzene ring, exhibits a blocking effect approximately 100 times weaker than tetracaine. We hypothesize that: 1) the binding site of the inactivating enhancer is located within the Na+ osmotic pathway; 2) external Na+ ions antagonize the blocking effect of the inactivating enhancer through electrostatic repulsion; 3) the 4-butylamino functional group on the benzene ring is crucial for both blocking and enhancing inactivation; and 4) overlapping binding sites for inactivating enhancers and opening channel blockers may exist within the Na+ pores.
Objective: To investigate the response of different sodium channel subtypes to general anesthetics, we examined the effect of pentobarbital (an analogue of thiopental sodium) on individual sodium channels in human skeletal muscle and compared it with existing data from human brain and ventricular muscle channels. Methods: Human skeletal muscle sodium channel preparations were integrated into a planar lipid bilayer, and the steady-state behavior of individual sodium channels and their response to pentobarbital were examined in the presence of the sodium channel activator tetrodotoxin. Single-channel currents were recorded before and after the addition of pentobarbital (0.34–1.34 mM). Results: In a symmetrical 500 mM NaCl solution, the mean single-channel conductance of human skeletal muscle sodium channels was 21.0 ± 0.6 pS, with a channel open fraction of 0.96 ± 0.04. The activation midpoint potential was -96.2 ± 1.6 mV. Extracellular tetrodotoxin blocked the channel at 0 mV with a half-maximal concentration (K1/2) of 60 nM. Pentobarbital reduced the time-mean conductance of individual skeletal muscle sodium channels in a concentration-dependent manner (IC50 = 0.66 mM). The steady-state activation potential shifted to a more negative potential (-16.7 mV with 0.67 mM pentobarbital). Conclusion: In a planar lipid bilayer system, some electrophysiological properties of skeletal muscle sodium channels differ significantly from those of sodium channels in cardiac or central nervous tissue. In contrast to control data, these different human sodium channel subtypes exhibited the same qualitative and quantitative responses to the general anesthetic pentobarbital. The impact of these effects on overall anesthetic efficacy depends on the role of each channel within its neuronal network, but inhibition of central and peripheral sodium channels may enhance general anesthetic effects. We investigated the effects of procainamide on brucellus toxin (BTX)-activated sodium channels in bovine heart and mouse skeletal muscle. When applied intracellularly, procainamide induced rapid open-channel blockade. We estimated the rate constant using amplitude distribution analysis. Membrane depolarization increased the blockade rate and slowed the release of the blockade. The rate constants of cardiac and skeletal muscle channels were similar in both amplitude and voltage dependence. Qualitatively, this blockade was similar to the rapid open-channel blockade induced by lidocaine, but procainamide was approximately seven-fold less potent. Molecular modeling suggests that the difference in potency between procainamide and lidocaine may stem from the relative orientation of their aromatic rings or differences in the structure of their aromatic amine bonds. For cardiac channels, procainamide reduced the frequency of channel transition to a long-lived closed state, which is characterized by inactivation. The mean duration of the kinetically determined closed state was unaffected. The degree of inhibition of rapid and slow closure was positively correlated. Endogenous application of the lidocaine derivative QX-314 (also a rapid blocker) produced a similar effect. Therefore, the binding of the drug to the rapid blocking site appears to inhibit the inactivation of BTX-activated cardiac channels. This study aimed to investigate the modification characteristics of Na+ channels in cardiomyocytes by poison dart frog toxin (BTX), including changes in channel gating and kinetics, and sensitivity to local anesthetic blockade. We measured Na+ currents in guinea pig cardiomyocytes using a whole-cell patch-clamp technique. To maintain good voltage control, we reduced extracellular Na+ concentration and temperature (5–10 mM, 17 °C). Our findings indicate that: 1) BTX alters the homeostatic (non-inactivated) composition of cardiac sodium channels, resulting in a significant homeostatic (non-inactivated) component within the channels; 2) BTX modification of cardiac sodium channels shifts the activation potential towards a more negative direction and reduces the maximum sodium channel blockade threshold (gNa) and sodium ion selectivity; 3) BTX binding to its receptors on cardiac sodium channels reduces the affinity of local anesthetics for their binding sites; 4) BTX-modified channels exhibit use-dependent blockade of local anesthetics. The reduced blocking efficacy of local anesthetics on BTX-modified sodium channels is likely due to allosteric interactions between BTX and local anesthetics at their sodium channel binding sites. We observed that use-dependent blockade of local anesthetics persists in BTX-modified sodium channels, suggesting that this additional form of blockade can occur even in the absence of an inactivated state. Therefore, under these conditions, the occurrence of use-dependent blockade appears to depend primarily on the binding of local anesthetics to activated Na+ channels. For more complete data on interactions of batroxotoxins (6 in total), please visit the HSDB record page.

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Non-human toxicity values
Intravenous LD50 in mice: 0.002 mg/kg
Dermal LD50 in mice: 2 μg/kg

[1]. Structure of batrachotoxin, a steroidal alkaloid from the Colombian arrow poison frog, Phyllobates aurotaenia, and partial synthesis of batrachotoxin and its analogs and homologs. Journal of the American Chemical Society 91.14 (1969): 3931-3938.

Batrachotoxin has been reported in the golden-banded frog (Phyllobates aurotaenia), and relevant data are available. Batrachotoxin is a potent sodium channel activator, found in frog skin extracts. Mechanism of Action: Batrachotoxin is a potent regulator of voltage-gated sodium channels, causing irreversible depolarization of nerves and muscles, tremors, arrhythmias, and ultimately heart failure. Since its discovery, field researchers have also reported numbness after skin contact with the toxin. Intrigued by this phenomenon, we investigated the effects of batrachotoxin on the voltage-gated sodium channel Nav1.8, considered a key player in nociception. We found that batrachotoxin significantly modulates this channel: the inactivation process is severely altered, the voltage dependence of activation shifts to a more negative membrane potential, causing Nav1.8 to open at a more negative membrane potential, and ion selectivity is also altered.
Bantox (BTX), derived from the genus Phyllobates of South American frogs, irreversibly activates voltage-gated sodium channels. Previous studies have shown that phenylalanine residues located near the midpoint of the transmembrane segment IVS6 within the channel liner are key factors determining the channel's sensitivity to BTX. In this study, we introduced a series of mutations at this site in the Na(v)1.3 sodium channel, expressing both wild-type and mutant channels in Xenopus oocytes, and examined their sensitivity to BTX using voltage-clamp recording. We found that substitutions of alanine or isoleucine significantly reduced the channel's sensitivity to the toxin, while substitutions of cysteine, tyrosine, or tryptophan only slightly reduced the toxin's effect. These data suggest the presence of electrostatic ligand-receptor interactions at this site, potentially involving charged tertiary amines on BTX. Subsequently, we used a mutant channel with moderate toxin sensitivity (mutant F1710C) to investigate the characterization of the toxin-receptor response in more detail. Unlike wild-type channels that bind BTX almost irreversibly, the toxin dissociates rapidly from mutant channels, but only when the channel is open, not when it is closed. These data suggest that the closed activation gate traps the bound toxin. Although BTX dissociation requires channel activation, paradoxically, strong membrane depolarization slows the dissociation rate, suggesting that the toxin binding response is also influenced by other state-dependent and/or electrostatic factors. We propose that BTX is transported to its receptor via the cytoplasmic end of an open ion channel pore, similar to quaternary ammonium salt local anesthetics (e.g., QX314). We covalently labeled purified and recombinant rat brain sodium ion channels with photoreactive radiolabeled batroxotoxin derivatives to locate the binding site of batroxotoxin (a lipid-soluble neurotoxin acting on the 2 site of sodium ion channel receptors). In the presence of Ptychodiscus brevis toxin 1 and the pyrethroid insecticide RU51049 (a positive allosteric enhancer for batroxotoxin binding), a protein with an apparent molecular weight of 240 kDa (corresponding to the α subunit of a sodium ion channel) was specifically covalently labeled. The α subunit region specifically photolabeled by the photoreactive batroxotoxin derivative was identified by antibody localization of the proteolytic fragment. Even after thorough trypsin digestion, antipeptide antibodies recognizing the amino acid sequence adjacent to the IS6 transmembrane segment of the sodium ion channel still immunoprecipitated up to 70% of the labeled peptide. More thorough digestion analysis with trypsin or V8 protease revealed that the toxin receptor site of the poison dart frog is partially composed of a portion of domain I. A photolabeled 7.3 kDa peptide with specific immunoprecipitation was identified, containing the transmembrane S6 region of domain I, thereby confining the labeling site to residues Asn-388 to Glu-429 (if digested completely by V8 protease) or Leu-380 to Glu-429 (if digested incompletely). These results indicate that the S6 transmembrane region of the sodium channel α-subunit I domain is an important component of the dart poison toxin receptor site.
The steroidal neurotoxin (-)-dart poison toxin is a potent agonist of voltage-gated sodium ion channels (NaV). This paper reports the concise asymmetric synthesis of the natural (-) and non-natural (+) enantiomers of dart poison toxin, as well as the synthesis of two enantiomers of the C-20 benzoate-modified derivative. Electrophysiological characterization of these molecules against the NaV isoforms revealed that the non-natural toxin enantiomers are reversible antagonists of channel function, with activity significantly different from that of (-)-dart poison toxin. Protein mutagenesis experiments revealed that these enantiomers share a common binding site within the NaV pore lumen. These findings inspired and propelled subsequent research aimed at elucidating how small molecules targeting the channel pore regulate the dynamics of sodium ion channels (NaV).
Betatoxin (BTX): A neurotoxin that activates sodium ion channels; a cardiotoxin similar to digoxin, capable of causing atrial fibrillation and cardiac arrest. /Excerpt from table/

C31H42N2O6

538.67

538.304

23509-16-2

6324647

Noncrystal

1.34g/cm3

744ºC at 760mmHg

403.8ºC

3E-23mmHg at 25°C

1.642

3.49

3

7

4

39

1140

8

CC1=CNC(=C1C(=O)O[C@@H](C)C2=CC[C@@]34[C@@]2(C[C@H]([C@@]56C3=CC[C@H]7[C@@]5(CC[C@](C7)(O6)O)C)O)CN(CCO4)C)C

ISNYUQWBWALXEY-OMIQOYQYSA-N

InChI=1S/C31H42N2O6/c1-18-16-32-19(2)25(18)26(35)38-20(3)22-8-9-30-23-7-6-21-14-29(36)11-10-27(21,4)31(23,39-29)24(34)15-28(22,30)17-33(5)12-13-37-30/h7-8,16,20-21,24,32,34,36H,6,9-15,17H2,1-5H3/t20-,21+,24+,27-,28-,29+,30-,31-/m0/s1

[(1S)-1-[(1R,5R,6S,9R,11S,12R,14R)-9,12-dihydroxy-6,16-dimethyl-10,19-dioxa-16-azahexacyclo[12.5.3.15,9.01,14.02,11.06,11]tricosa-2,21-dien-22-yl]ethyl] 2,4-dimethyl-1H-pyrrole-3-carboxylate

BTX

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.)