Toxicity Summary
IDENTIFICATION AND USE: Batrachotoxins are neurotoxic steroidal alkaloids first isolated from a Colombian poison-dart frog and later found in certain passerine birds of New Guinea. Neither vertebrate group is thought to produce the toxins de novo, but instead they likely sequester them from dietary sources. HUMAN STUDIES: Batrachotoxin is a potent modulator of voltage-gated sodium channels, leading to irreversible depolarization of nerves and muscles, fibrillation, arrhythmias and eventually cardiac failure. Since its discovery, field researchers also reported numbness after their skin came into contact with this toxin. ANIMAL STUDIES: Levels of batrachotoxin tend to be reduced when P. terribilis is maintained in captivity, but even after being confined for up to 6 years, these frogs were still at least five times more toxic than other Phyllobates species used by natives for poisoning blowgun darts. Batrachotoxin was not detectable in F1 progeny reared to maturity in captivity. Nerve and muscle preparations from wild-caught frogs and from the nontoxic F1 frogs were both insensitive to batrachotoxin. The regulatory site controlling sodium-channel activation and permeability appears to have been minimally altered to prevent interaction with batrachotoxin, but is still sensitive to other sodium conductance activators (veratridine, grayanotoxin) to which the frogs are not exposed naturally.

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Interactions
Two distinct types of local anesthetics (LAs) have previously been found to block batrachotoxin (BTX)-modified Na+ channels: type 1 LAs such as cocaine and bupivacaine interact preferentially with open channels, whereas type 2 LAs, such as benzocaine and tricaine, with inactivated channels. Herein, we describe our studies of a third type of LA, represented by tetracaine as a dual blocker that binds strongly with closed channels but also binds to a lesser extent with open channels when the membrane is depolarized. Enhanced inactivation of BTX-modified Na+ channels by tetracaine was determined by steady-state inactivation measurement and by the dose-response curve. The 50% inhibitory concentration (IC50) was estimated to be 5.2 microM at -70 mV, where steady-state inactivation was maximal, with a Hill coefficient of 0.98 suggesting that one tetracaine molecule binds with one inactivated channel. Tetracaine also interacted efficiently with Na+ channels when the membrane was depolarized; the IC50 was estimated to be 39.5 microM at +50 mV with a Hill coefficient of 0.94. Unexpectedly, charged tetracaine was found to be the primary active form in the blocking of inactivated channels. In addition, external Na+ ions appeared to antagonize the tetracaine block of inactivated channels. Consistent with these results, N-butyl tetracaine quaternary ammonium, a permanently charged tetracaine derivative, remained a strong inactivation enhancer. Another derivative of tetracaine, 2-(di-methylamino) ethyl benzoate, which lacked a 4-butylamino functional group on the phenyl ring, elicited block that was approximately 100-fold weaker than that of tetracaine. We surmise that 1) the binding site for inactivation enhancers is within the Na+ permeation pathway, 2) external Na+ ions antagonize the block of inactivation enhancers by electrostatic repulsion, 3) the 4-butylamino functional group on the phenyl ring is critical for block and for the enhancement of inactivation, and 4) there are probably overlapping binding sites for both inactivation enhancers and open-channel blockers within the Na+ pore
PURPOSE: To investigate the response to general anesthetics of different sodium-channel subtypes, we examined the effects of pentobarbital, a close thiopental analogue, on single sodium channels from human skeletal muscle and compared them to existing data from human brain and human ventricular muscle channels. METHODS: Sodium channels from a preparation of human skeletal muscle were incorporated into planar lipid bilayers, and the steady-state behavior of single sodium channels and their response to pentobarbital was examined in the presence of batrachotoxin, a sodium-channel activator. Single-channel currents were recorded before and after the addition of pentobarbital (0.34-1.34 mM). RESULTS: In symmetrical 500 mM NaCl, human skeletal muscle sodium channels had an averaged single-channel conductance of 21.0 +/- 0.6 pS, and the channel fractional open time was 0.96 +/- 0.04. The activation midpoint potential was -96.2 +/- 1.6 mV. Extracellular tetrodotoxin blocked the channel with a half-maximal concentration (k1/2) of 60 nM at 0 mV. Pentobarbital reduced the time-averaged conductance of single skeletal muscle sodium channels in a concentration-dependent manner (inhibitory concentration 50% [IC50] = 0.66 mM). The steady-state activation was shifted to more hyperpolarized potentials (-16.7 mV at 0.67 mM pentobarbital). CONCLUSION: In the planar lipid bilayer system, skeletal muscle sodium channels have some electrophysiological properties that are significantly different compared with those of sodium channels from cardiac or from central nervous tissue. In contrast to the control data, these different human sodium channel subtypes showed the same qualitative and quantitative response to the general anesthetic pentobarbital. The implication of these effects for overall anesthesia will depend on the role the individual channels play within their neuronal networks, but suppression of both central nervous system and peripheral sodium channels may add to general anesthetic effects
We have investigated the action of procainamide on batrachotoxin (BTX)-activated sodium channels from bovine heart and rat skeletal muscle. When applied to the intracellular side, procainamide induced rapid, open-channel block. We estimated rate constants using amplitude distribution analysis. Membrane depolarization increased the blocking rate and slowed unblock. The rate constants were similar in both magnitude and voltage dependence for cardiac and skeletal muscle channels. Qualitatively, this block resembled the fast open-channel block by lidocaine, but procainamide was about sevenfold less potent. Molecular modeling suggests that the difference in potency between procainamide and lidocaine might arise from the relative orientation of their aromatic rings, or from differences in the structure of the aryl-amine link. For the cardiac channels, procainamide reduced the frequency of transitions to a long-lived closed state which shows features characteristic of inactivation. Mean durations of kinetically identified closed states were not affected. The degree of fast block and of inhibition of the slow closures were correlated. Internally applied QX-314, a lidocaine derivative and also a fast blocker, produced a similar effect. Thus, drug binding to the fast blocking site appears to inhibit inactivation in BTX-activated cardiac channels.
The purpose of the present study was to examine the characteristics of Na+ channel modification by batrachotoxin (BTX) in cardiac cells, including changes in channel gating and kinetics as well as susceptibility to block by local anesthetic agents. We used the whole cell configuration of the patch clamp technique to measure Na+ current in guinea pig myocytes. Extracellular Na+ concentration and temperature were lowered (5-10 mM, 17 degrees C) in order to maintain good voltage control. Our results demonstrated that 1) BTX modifies cardiac INa, causing a substantial steady-state (noninactivating) component of INa, 2) modification of cardiac Na+ channels by BTX shifts activation to more negative potentials and reduces both maximal gNa and selectivity for Na+; 3) binding of BTX to its receptor in the cardiac Na+ channel reduces the affinity of local anesthetics for their binding site; and 4) BTX-modified channels show use-dependent block by local anesthetics. The reduced blocking potency of local anesthetics for BTX-modified Na+ channels probably results from an allosteric interaction between BTX and local anesthetics for their respective binding sites in the Na+ channel. Our observations that use-dependent block by local anesthetics persists in BTX-modified Na+ channels suggest that this form of extra block can occur in the virtual absence of the inactivated state. Thus, the development of use-dependent block appears to rely primarily on local anesthetic binding to activated Na+ channels under these conditions.
For more Interactions (Complete) data for Batrachotoxin (6 total), please visit the HSDB record page.

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Non-Human Toxicity Values
LD50 Mice iv 0.002 mg/kg
LD50 Mice dermal 2 ug/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 Phyllobates aurotaenia with data available.
Batrachotoxin is batrachotoxins are potent activators of sodium channels identified in extract of frog skins.

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Mechanism of Action
Batrachotoxin is a potent modulator of voltage-gated sodium channels, leading to irreversible depolarization of nerves and muscles, fibrillation, arrhythmias and eventually cardiac failure. Since its discovery, field researchers also reported numbness after their skin came into contact with this toxin. Intrigued by this phenomenon, we determined the effect of batrachotoxin on the voltage-gated sodium channel Nav1.8, which is considered to be a key player in nociception. As a result, we discovered that batrachotoxin profoundly modulates this channel: the inactivation process is severely altered, the voltage-dependence of activation is shifted towards more hyperpolarized potentials resulting in the opening of Nav1.8 at more negative membrane potentials and the ion selectivity is modified.
Batrachotoxin (BTX), from South American frogs of the genus Phyllobates, irreversibly activates voltage-gated sodium channels. Previous work demonstrated that a phenylalanine residue approximately halfway through pore-lining transmembrane segment IVS6 is a critical determinant of channel sensitivity to BTX. In this study, we introduced a series of mutations at this site in the Na(v)1.3 sodium channel, expressed wild-type and mutant channels in Xenopus laevis oocytes, and examined their sensitivity to BTX using voltage clamp recording. We found that substitution of either alanine or isoleucine strongly reduced channel sensitivity to toxin, whereas cysteine, tyrosine, or tryptophan decreased toxin action only modestly. These data suggest an electrostatic ligand-receptor interaction at this site, possibly involving a charged tertiary amine on BTX. We then used a mutant channel (mutant F1710C) with intermediate toxin sensitivity to examine the properties of the toxin-receptor reaction in more detail. In contrast to wild-type channels, which bind BTX almost irreversibly, toxin dissociation from mutant channels was rapid, but only when the channels were open, not when they were closed. These data suggest the closed activation gate trapped bound toxin. Although BTX dissociation required channel activation, it was, paradoxically, slowed by strong membrane depolarization, suggesting additional state-dependent and/or electrostatic influences on the toxin binding reaction. We propose that BTX moves to and from its receptor through the cytoplasmic end of the open ion-conducting pore, in a manner similar to that of quaternary local anesthetics like QX314.
The binding site for batrachotoxin, a lipid-soluble neurotoxin acting at Na+ channel receptor site 2, was localized using a photoreactive radiolabeled batrachotoxin derivative to covalently label purified and reconstituted rat brain Na+ channels. In the presence of the brevetoxin 1 from Ptychodiscus brevis and the pyrethroid RU51049, positive allosteric enhancers of batrachotoxin binding, a protein with an apparent molecular mass of 240 kDa corresponding to the Na+ channel alpha subunit was specifically covalently labeled. The region of the alpha subunit specifically photolabeled by the photoreactive batrachotoxin derivative was identified by antibody mapping of proteolytic fragments. Even after extensive trypsinization, and anti-peptide antibody recognizing an amino acid sequence adjacent to Na+ channel transmembrane segment IS6 was able to immunoprecipitate up to 70% of the labeled peptides. Analysis of a more complete digestion with trypsin or V8 protease indicated that the batrachotoxin receptor site is formed in part by a portion of domain I. The identification of a specifically immunoprecipitated photolabeled 7.3-kDa peptide containing transmembrane segment S6 from domain I restricted the site of labeling to residues Asn-388 to Glu-429 if V8 protease digestion was complete or Leu-380 to Glu-429 if digestion was incomplete. These results implicate the S6 transmembrane region of domain I of the Na+ channel alpha subunit as an important component of the batrachotoxin receptor site.
The steroidal neurotoxin (-)-batrachotoxin functions as a potent agonist of voltage-gated sodium ion channels (NaVs). Here we report concise asymmetric syntheses of the natural (-) and non-natural (+) antipodes of batrachotoxin, as well both enantiomers of a C-20 benzoate-modified derivative. Electrophysiological characterization of these molecules against NaV subtypes establishes the non-natural toxin enantiomer as a reversible antagonist of channel function, markedly different in activity from (-)-batrachotoxin. Protein mutagenesis experiments implicate a shared binding side for the enantiomers in the inner pore cavity of NaV. These findings motivate and enable subsequent studies aimed at revealing how small molecules that target the channel inner pore modulate NaV dynamics.
Batrachotoxins (BTX): Neurotoxin, activates sodium ion channels, digitalis (digitoxin)-like cardiotoxin, causes fibrillation and cardiac arrest. /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.)