Voltage gated sodium channels

Tetrodotoxin is a neurotoxin with potential analgesic activity. Tetrodotoxin binds to the pores of fast voltage-gated fast sodium channels in nerve cell membranes, inhibiting nerve action potentials and blocking nerve transmission.

The addition of veratridine, 2 min after anoxia, caused a reduction of lactate synthesis which was completely blocked about 30 min after veratridine was applied. The application of tetrodotoxin (TTX), prior to veratridine, prevented the veratridine-induced inhibition of lactate synthesis, whereas (±)-kavain was less effective than TTX (Fig. 2(A)). Despite the linearity of lactate production of untreated vesicles (Fig. 2(A)), the ATP content declined continuously with a half life (τ) of 14.5 min (Table 1) upon anoxia. After 60 min of anoxia, ATP was reduced to 21.0% of its initial value (2.85 ± 0.16 nmol ATP/mg protein, n = 6) determined under oxygenated conditions. An additional stimulation of anoxic vesicles by veratridine accelerated the reduction in ATP which declined three times faster than the ATP content of untreated vesicles (Table 1). An addition of TTX before veratridine (Fig. 2(B)) did not only prevent the veratridine effect but compared with untreated vesicles, TTX also reduced the velocity of ATP decline (Table 1) and improved the ATP content during anoxia (Fig. 2(B)). (±)-Kavain also suppressed the veratridine effect on ATP, but with less efficacy than TTX (Fig. 2(B)). According to Table 1, the τ of ATP decline and ATP content, the latter determined 60 min after anoxia, were correlated in the following order: [veratridine + TTX] > [control] > [veratridine + (±)-kavain] > [veratridine]. If vesicles were stimulated with 40 mmol/l KCl instead of veratridine, neither inhibition of lactate synthesis (Fig. 3(A)) nor any effect on ATP-content could be detected (Fig. 3(B)). It should be emphasized that in contrast to the results obtained with veratridine-treated vesicles (Fig. 2(B)), the application of TTX to KCl-depolarized vesicles failed to improve the ATP-content compared with the controls (Fig. 3(B)). Like TTX, (±)-kavain was without any effect on lactate synthesis (Fig. 3(A)), the velocity of ATP decline and the final ATP content determined at the end of anoxia (Fig. 3(B)). [1]

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Since only veratridine blocked lactate synthesis after prolonged anoxia and speeded up the decline in ATP content, [Na+]i, [Ca2+]i and lactate production were followed continuously during short-term anoxia and veratridine stimulation to determine whether Na+ overload influences lactate synthesis directly. As shown in Fig. 4(A), [Na+]i increased persistently after onset of anoxia and basal [Na+]i increased from 18 ± 2 to 41 ± 7 mmol/l Na+ detected 380 sec after anoxia. An additional stimulation of anoxic vesicles with veratridine increased [Na+]i immediately, then tended to level off to 119 ± 21 mmol/l Na+ (n = 6, Fig. 4(A)). The application of tetrodotoxin (TTX) and (±)-kavain before anoxia either diminished or prevented the anoxia- and veratridine-induced increases in [Na+]i, respectively. However, both compounds failed to block the increase of [Na+]i completely (Fig. 4(A)). To estimate the inhibition of anoxia-induced increases in [Na+]i by TTX and (±)-kavain, the differences between basal and anoxia-induced [Na+]i determined 20 sec before and 380 sec after onset of anoxia were calculated and expressed as Δ[Na+]i. The Δ[Na+]i of untreated vesicles (22.1 ± 6.1 mmol/l Na+, n = 6) were suppressed by TTX and (±)-kavain to 77% (17 ± 5 mmol/l Na+, n = 6) and 59% (13 ± 6 mmol/l Na+, n = 6) of untreated vesicles, respectively (Fig. 4(A)). [1]

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Regarding [Ca2+]i measurements, basal [Ca2+]i of 581 ± 86 nmol/l Ca2+, as detected 20 sec before anoxia, increased continuously to 856 ± 87 nmol/l Ca2+ (n = 6) after 380 sec of anoxia (Fig. 4(B)). The addition of veratridine to anoxic vesicles provoked a rapid increase in [Ca2+]i which, in contrast to [Na+]i (Fig. 4(A)), failed to level off but increased linearly (Fig. 4(B)) with a rate of 355 ± 126 nmol Ca2+/min/mg protein (n = 6). The application of tetrodotoxin (TTX) or (±)-kavain before anoxia reduced the anoxia- and veratridine-induced enhancement of [Ca2+]i but, as already observed with [Na+]i, both compounds did not prevent the increase in [Ca2+]i completely (Fig. 4(B)). Considering the efficacy of TTX and (±)-kavain on anoxia-enhanced [Ca2+]i, Δ[Ca2+]i was calculated by analogy to Δ[Na+]i, as described above. TTX and (±)-kavain diminished the Δ[Ca2+]i of untreated vesicles (275 ± 35 nmol/l Ca2+, n = 6) to 41% (113 ± 13 nmol/l Ca2+, n = 6) and 48% (133 ± 35 nmol/l Ca2+, n = 6), respectively. [1]

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In order to compare the time courses of [Na+]i and [Ca2+]i increase with lactate synthesis, lactate production was monitored continuously by the fluorometric method. As shown in Fig. 5, the rate of basal lactate synthesis of non-stimulated vesicles was enhanced 4.2-fold from 3.5 ± 1.4 to 14.6 ± 1.5 nmol lactate/min/mg protein upon anoxia (Table 1). These rates, calculated by linear regression, are 1.2–1.5 times larger than the rates calculated according to the method of spectrophotometric lactate determination. The cause for this difference remained unclear and might depend on the different incubation procedures. However, the addition of veratridine 240 sec before anoxia doubled lactate production (Fig. 5, trace 4) is suggested to be a result of enhanced energy demand due to an activation of Na+/K+-ATPase by the induced Na+ influx, since tetrodotoxin (TTX) and (±)-kavain, both applied 120 sec prior to veratridine, prevented this stimulation (Fig. 5). Regarding anaerobic lactate synthesis, neither veratridine nor the pre-application of TTX and (±)-kavain affected the rates of lactate production (Fig. 5, Table 1) during 500 sec of anoxia, an incubation time which was sufficient to enhance [Na+]i and [Ca2+]i to more than 120 mmol/l Na+ and 3 μmol/l Ca2+ (Fig. 4(A,B)).

Spectrophotometric lactate determination [1]
The vesicle pellet was resuspended in 3 ml incubation buffer (125 mmol/l NaCl, 3.5 mmol/l KCl, 1.2 mmol/l CaCl2, 1.2 mmol/l MgCl2, 25 mmol/l NaHCO3, 10 mmol/l glucose) equilibrated with 95% O2 and 5% CO2 to obtain final protein concentrations of 3 mg/ml. The suspension was transferred into a self-made, closed chamber surrounded by a water jacket to maintain an incubation temperature of 37°C. Additionally, the incubation chamber was equipped with two gas taps for in- and out-streaming gas, allowing a surface equilibration. The O2 content of the suspension, expressed as % O2 saturation, was monitored continuously with a built-in, Clark-type, oxygen electrode, which was calibrated with sodium dithionite (Hitchman, 1978). One hundred percent O2 saturation was adjusted by equilibration of incubation buffer with O2 for 45 min at 37°C and 0.0% O2 saturation was attained by the addition of 500 mmol/l sodium dithionite, which traps dissolved O2 according to the reaction: 2Na2S2O4+2H2O+3H2O→4NaHSO4. After bubbling the suspension for 15 min with 95% O2 and 5% CO2 at 37°C, it was equilibrated with 95% N2 and 5% CO2 and the vesicles were allowed to respire until the medium was depleted of oxygen (0.0% O2 saturation). This point was defined as onset of anoxia. Lactate was determined enzymatically by a commercially available test kit according to the instructions of the supplier. Samples of vesicles, taken as indicated, were immediately frozen in liquid nitrogen and stored at −20°C until performance of the lactate assay. l-Lactate was determined spectrophotometrically (Noll, 1984) by the generation of NADH due to the oxidation of lactate to pyruvate, catalysed by lactate dehydrogenase (LDH, EC 1.1.1.27).

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ATP determination [1]
Twenty microlitres of the vesicle suspension with a protein content of about 3 mg/ml were mixed with 200 μl precooled 1.0 mol/l perchloric acid, 50 mmol/l EDTA at 0°C for 60 sec. The suspension was adjusted to pH 7.5–8.0 by the addition of 316 μl 1.0 mol/l KOH and 180 μl 0.1 mol/l HEPES, 10 mmol/l MgCl2 (pH 7.75, 25°C) and centrifuged at 14 000g for 10 min. Five-hundred microlitres of the supernatant was mixed with 500 μl 0.1 mol/l HEPES, 10 mmol/l MgCl2 (pH 7.75, 25°C) and the ATP content was detected by the luciferase-bioluminescence method employing the kit HS II and the luminometer LB 9502 (Berthold GmbH, Bad Wildbad, Germany). The ATP content was calculated according to a calibration curve, using different ATP concentrations as standards.

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Fluorometric lactate determination [1]
Continuous detection of liberated l-lactate during short-term anoxia was performed enzymatically employing LDH (EC 1.1.1.27) and 3-acetylpyridineadeninedinucleotide (APAD) as an analogue of NAD (Kaplan and Ciotti, 1956) according to the reaction: l-lactate+ APAD ↫ pyruvate+APADH. The generation of APADH was fluorometrically detected at excitation and emission wavelengths of 410 nm and 490 nm, respectively, taking bandpass slits of 20 nm for both monochromators. For fluorescence measurement, each vesicle pellet was resuspended in 3 ml incubation buffer to obtain a final protein concentration of 3 mg/ml. The suspension was transferred to a temperature-controlled, stirred cuvette located in a spectrofluorometer. Additionally, LDH and APAD were added to obtain final concentrations of 50 units/ml LDH and 5 mmol/l APAD. Afterwards, the cuvette was sealed with a cap equipped with two gas taps for in- and out-streaming gas and a built-in, Clark-type, oxygen electrode allowing continuous oxygen detection. Anoxic conditions were induced by surface equilibration of the suspension with 95% N2 and 5% CO2. The amount of lactate released from vesicles was calculated according to a calibration curve performed with a vesicle suspension (3 mg/ml protein) and different l-lactate standards.

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Intravesicular NADH ([NADH]i) [1]
Three millilitres of vesicle suspension (3 mg/ml protein) were transferred to a cuvette and incubated as described for fluorometric lactate determination, except for the addition of LDH and APAD. The increase in [NADH]i was followed by its fluorescence, determined at excitation and emission wavelengths of 340 nm and 460 nm, respectively, taking bandpass slits of 20 nm for both monochromators.

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Loading with SBFI-AM and FURA-2-AM [1]
[Na+]i and [Ca2+]i were determined by the ratio fluorescence method employing the acetoxymethyl esters (AM) of SBFI and FURA-2 as Na+- and Ca2+-sensitive fluorophores, respectively (Minta and Tsien, 1989; Grynkiewicz et al., 1985). Considering [Ca2+]i measurements, 80 μl of 1 mmol/l FURA-2-AM, dissolved in dimethylsulphoxide (DMSO), was added to 8 ml suspension with a protein content of about 4.5 mg/ml, to obtain final concentrations of 1% (v/v) DMSO and 10 μmol/l FURA-2-AM. In the case of [Na+]i determination, 66 μl of 2 mmol/l SBFI-AM and 22 μl 20% (w/v) pluronic F127, both dissolved in DMSO, were added to the suspension, to obtain final concentrations of 16.5 μmol/l SBR-AM, 0.055% (w/v) pluronic F127 and 1.1% (v/v) DMSO. The suspensions were incubated for 30 min at room temperature and subsequently washed three times with incubation buffer by centrifugation (5000g, 5 min) to remove unhydrolysed dye. Finally, the last pellet was resuspended in 12 ml incubation buffer, and the suspension was divided into portions of 9 mg protein. After centrifugation (5000g, 5 min) the pellets were stored on ice until measurement of fluorescence.

Absorption, Distribution and Excretion
In 1999, 23 specimens of the genus Polypedates were collected from two sites in Bangladesh (Mymensing and Barisal), and their toxicity scores and toxin components were determined. Of all tissues, only the skin of the Mymensing specimens showed toxicity in mouse tests, with toxicity scores ranging from 31 to 923 μg/g. The toxin isolated from the skin was identified as tetrodotoxin, a toxin component, by high-performance liquid chromatography, electrospray ionization time-of-flight mass spectrometry, and proton nuclear magnetic resonance analysis. Tetrodotoxin (TTX) and its analogues (TTXs) are widely distributed in marine and terrestrial animals and can cause dangerous poisoning. These highly toxic toxins are also pathogenic factors in pufferfish poisoning. Tetrodotoxin (TTX), dehydrated tetrodotoxin, 11-deoxytetrodotoxin, and trimeoxytetrodotoxin were determined in tissues isolated from the Bangladeshi pufferfish (Takifugu oblongus). Tetrodotoxin (TTX) was mainly found in the skin, muscle, and liver, while tripeoxytetrodotoxin was mainly found in the ovaries. The toxicity of each tissue was determined using a mouse bioassay. To investigate the relationship between tetrodotoxin toxicity and the distribution of tetrodotoxin-producing bacteria, we isolated bacteria from various organs (ovaries, liver, intestines, and gallbladder) of the red-finned pufferfish (Fugu rubripes) collected from the Bohai Sea, China, and screened their ability to produce tetrodotoxin (TTX). Of the 36 isolated strains, 20 were able to produce TTX in vitro. The number and toxicity of TTX-producing strains were higher in organs with higher toxicity, such as the ovaries and liver. Based on morphological observation, physiological and biochemical characteristics, and DNA G+C content, most TTX-producing strains were identified as Bacillus (19 strains) and Actinomyces (1 strain). The purified toxin was identified as TTX by high-performance liquid chromatography, thin-layer chromatography, and electrospray ionization mass spectrometry. Our results indicate that TTX-producing bacteria are closely related to pufferfish poisoning. Further research is needed to elucidate the synthetic mechanism of tetrodotoxin (TTX) and its role in bacteria.
Differential centrifugation was used to separate pufferfish liver homogenate into hemocyte, nucleus, mitochondria, microsomes, and cytosol components. High-performance liquid chromatography (HPLC) and liquid chromatography-fast atomic bombardment mass spectrometry (LC-FABMS) analysis showed that tetrodotoxin was the major toxic component in each component. These results reveal that tetrodotoxin is widely distributed in organelles of hepatocytes, but is mainly present in the cytosol component.
More data on the absorption, distribution, and excretion (ADEX) of tetrodotoxins (9 in total) can be found on the HSDB record page.
Metabolism/Metabolites
The metabolic source of tetrodotoxin is unclear. Algal sources have not been identified; until recently, tetrodotoxin was considered a host metabolite. However, recent reports indicate that various bacteria, including Vibrionaceae, Pseudomonas sp., and Photobacterium phosphoreum, can produce tetrodotoxin/dehydrated tetrodotoxin, suggesting that this toxin family may originate from bacteria. To investigate genes related to tetrodotoxin (TTX) biosynthesis or accumulation in pufferfish, we compared mRNA expression patterns in the livers of pufferfish (including Takifugu chrysops and Takifugu niphobles) carrying different concentrations of TTX and its derivatives using RAP RT-PCR. RAP RT-PCR yielded a 383 bp cDNA fragment whose transcript was expressed at higher levels in the livers of toxic pufferfish than in non-toxic pufferfish. Its deduced amino acid sequence is similar to fibrinogen-like proteins reported in other vertebrates. Northern blot analysis and rapid cDNA end amplification (RACE) revealed that the 383 bp cDNA fragment was composed of at least three fibrinogen-like protein (flp) genes, namely flp-1, flp-2, and flp-3. The relative mRNA levels of flp-1, flp-2, and flp-3 were linearly correlated with the toxicity of the livers of the two pufferfish species.

Toxicity Overview
Identification and Uses: Tetrodotoxin (TTX) is a solid. Since its channel-blocking effect was discovered in the early 1960s, TTX in inhaled aerosols has become an extremely common chemical tool in physiological and pharmacological laboratories. In recent years, TTX-tolerant sodium channels have been discovered in the nervous system and have attracted much attention due to their role in pain perception. It is now known that TTX is not produced by inhaled aerosols, but by bacteria and is distributed to various animals through the food chain. Human Studies: TTX is a lethal neurotoxin that selectively inhibits the Na(+) activation mechanism of nerve impulses without affecting K(+) ion permeability. TTX interferes with the transmission of nerve signals to muscles by blocking sodium channels. This leads to rapid weakness and paralysis of muscles (including respiratory muscles), which may eventually lead to respiratory arrest and death. Tetrodotoxin (TTX) poisoning can have a rapid onset (10 to 45 minutes) or a delayed onset (usually within 3 to 6 hours, but rarely longer than 3 to 6 hours). Death can occur within 20 minutes of exposure or up to 24 hours later; however, it usually occurs within the first 4 to 8 hours. Patients/victims who survive the acute poisoning phase within the first 24 hours usually recover without sequelae. Symptoms may last for several days, and recovery also takes several days. The first stage after ingestion of tetrodotoxin presents with numbness, tingling, and tingling of the lips and tongue (paresthesia), followed by paresthesia and numbness of the face and extremities, headache, lightheadedness, excessive sweating (hyperhidrosis), dizziness, drooling (salivation), nausea, vomiting, diarrhea, abdominal pain (upper abdominal pain), motor dysfunction, weakness (malaise), and difficulty speaking. The second stage presents with progressive paralysis, initially in the extremities, then in other parts of the body, and finally in the respiratory muscles; difficulty breathing or shortness of breath (tachypnea); cardiac arrhythmias; abnormally low blood pressure (hypotension); dilated pupils (fixed pupils); coma; seizures; respiratory arrest; and death. Several case reports describe food poisoning caused by tetrodotoxin (TTX). Most poisoning incidents occurred after consuming homemade pufferfish products, rather than commercially sourced pufferfish. In vitro experiments showed that TTX was not genotoxic in human lymphocytes, regardless of metabolic activation. Animal studies: Clinical symptoms and signs in dogs treated with intravenous TTX were similar to those of anticholinesterase poisoning. Studies found that oral TTX was approximately 50 times less toxic to mice than intraperitoneal injection, with later onset of death. In rats, serotonin levels in the brain significantly increased and peaked 4 hours after TTX administration. Levels of acetylcholine, histamine, and norepinephrine in the brain also significantly increased, but peaked after 6 hours. The effects of pufferfish gonadal extract were more pronounced and longer-lasting than those of skin extract. On the other hand, no significant changes in brain adrenaline levels were observed during the experiments. Intravenous injection of sublethal doses of tetrodotoxin (TTX) into male rabbits leads to perfusion failure, accompanied by lactic acidosis, hypoproteinemia, prolonged bleeding time, decreased red blood cell count, and decreased platelet count, resulting in shock. The severity of poisoning is directly proportional to the dose of tetrodotoxin. Autopsy studies revealed hemorrhage in the brain, liver, lungs, and diaphragm. Significant differences in susceptibility to TTX were observed among the five mouse strains tested. Both in vitro and in vivo experiments demonstrated that TTX is not genotoxic. Ecotoxicity studies: TTX and its analogues (TTXs) are widely distributed in marine and terrestrial animals and can cause dangerous poisoning. In addition to helping defend against predators, pufferfish's resistance to TTX allows them to selectively prey on organisms containing TTX. However, tetrodotoxin (TTX) does not protect Guam's flatworms from predators; instead, it is used to capture moving prey.

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Interactions
This study investigated the passive protective effect of a tetrodotoxin (TTX)-specific monoclonal antibody against lethal TTX challenge. The monoclonal antibody, T20G10, has an estimated affinity for TTX of approximately 10⁻⁹ M, exhibits approximately 50-fold lower reactivity to dehydrated tetrodotoxin, and is unresponsive to tetrodotoxin in a competitive immunoassay. T20G10 specifically inhibited TTX binding in an in vitro radioligand receptor binding assay but had no effect on the binding of tetrodotoxin to rat meningeal sodium channels. In a prophylactic study, mice were administered T20G10 via tail vein injection 30 minutes prior to challenge with intraperitoneal injection of TTX (10 μg/kg). Under these conditions, 100 μg of T20G10 protected 6/6 mice, while 50 μg of T20G10 protected 3/6 mice. The nonspecific control monoclonal antibody failed to prevent death. In a study simulating oral poisoning, a lethal dose of TTX was dissolved in phosphate buffer and skim milk powder via gavage. Six out of six mice that did not receive T20G10 treatment died within 25-35 minutes. However, intravenous injection of 500 μg of T20G10 via the tail vein 10-15 minutes after oral TTX prevented death in all six mice. Lower doses of monoclonal antibodies provided weaker protection. PMID: 8585093
In dogs 24 hours after coronary artery occlusion, lidocaine (4 mg/kg, intravenous) and tetrodotoxin (2 μg/kg, intravenous) both showed significant antiarrhythmic activity. When the dose was reduced by half, neither substance alone had any effect on arrhythmias, but when used in combination, cardiac rhythm was almost completely restored.
Frog toxin increases sodium uptake by synaptosomes. Veratrine also increases sodium uptake. Tetrodotoxin blocks the effects of the above toxins.
Denervated muscles in mice were removed after 5-6 days and incubated in 0.5% papain solution at 28-9°C for 5-8 minutes. This proteolytic treatment, by partially removing unstable surface proteins, eliminated the shielding effect of sodium ion channels, thereby restoring the sensitivity of receptors to tetrodotoxin after denervation injury.

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Non-human toxicity values
Oral LD50 in mice: 0.435 mg/kg Lewis, RJ Sr. (ed) Sax's Dangerous Properties of Industrial Materials. 11th Edition. Wiley-Interscience, Wiley & Sons, Inc. Hoboken, NJ. 2004., p. 1827
Intraperitoneal LD50 in mice: 0.008 mg/kg Lewis, RJ Sr. (ed) Sax's Dangerous Properties of Industrial Materials. 11th Edition. Wiley-Interscience, Wiley & Sons, Inc. Hoboken, NJ. 2004., p. 1827
Subcutaneous LD50 in mice: 0.008 mg/kg. Lewis, RJ Sr. (ed.) Sax's Dangerous Properties of Industrial Materials, 11th Edition. Wiley-Interscience, Wiley & Sons, Inc., Hoboken, NJ, 2004, p. 1827.
Intravenous LD50 in mice: 0.009 mg/kg. Lewis, RJ Sr. (ed.), Hazardous Properties of Materials for Saxe Industries, 11th ed. Wiley-Interscience, Wiley & Sons, Inc., Hoboken, NJ, 2004, p. 1827.

[1]. The protective action of tetrodotoxin and (±)-kavain on anaerobic glycolysis, ATP content and intracellular Na+and Ca2+of anoxic brain vesicles.Neuropharmacology, 1996, 35,1743.

Tetrodotoxin is an aminoperhydroquinazoline toxin primarily found in the liver and ovaries of tetradontidae fish, which are edible. The toxin causes paresthesia and paralysis by interfering with neuromuscular transmission. Wex Pharmaceuticals is investigating tetrodotoxin for the treatment of chronic and explosive pain in patients with advanced cancer, as well as for opioid dependence. Tetrodotoxin has also been reported in yellowfin pufferfish (Takifugu flavidus), redfin pufferfish (Takifugu rubripes), and several other organisms with relevant data. Tetrodotoxin is a neurotoxin with potential analgesic activity. It binds to the pores of rapidly voltage-gated sodium channels on nerve cell membranes, inhibiting nerve action potentials and blocking nerve conduction. Although tetrodotoxin is present in a variety of fish (e.g., pufferfish), salamanders, frogs, flatworms, and crabs, there is currently no known antidote. It is actually produced by Vibrio alginolyticus, Pseudomonas tetradontidae, and other Vibrio and Pseudomonas bacteria. Tetrodotoxin is an aminoperhydroquinazoline toxin found primarily in the liver and ovaries of edible tetrodotoxin fish. This toxin causes paresthesia and paralysis by interfering with neuromuscular transmission. Drug Indications: Used to treat chronic and explosive pain in patients with advanced cancer, and to treat opioid dependence. Mechanism of Action: Tetrodotoxin binds to site 1 of a fast-voltage-gated sodium channel located at the opening of extracellular pores. Binding of any molecule to this site temporarily inhibits the function of the ion channel. Tetrodotoxin and several cone snail toxins also bind to this site. The sodium current (I(Na)) of the mammalian heart is resistant to tetrodotoxin (TTX) due to the low affinity of the cardiac sodium channel (Na(v)) subtype Na(v)1.5 for TTX. To test whether this finding applies to other vertebrates, we examined the sensitivity of fish cardiac I(Na) to TTX and its molecular composition. Methods: We used molecular cloning and whole-cell patch-clamp techniques to investigate the composition of the α subunit of Na(v) in the heart of rainbow trout (Oncorhynchus mykiss) and the inhibitory effect of tetrodotoxin (TTX). The sodium ion current (I(Na)) in the rainbow trout heart was approximately 1000 times more sensitive to tetrodotoxin (TTX) than that in the mammalian heart (IC50 = 1.8–2 nM). It was generated by three sodium ion channel (Na(v)) α subunits, which were homologous to Na(v)1.4 in mammalian skeletal muscle, Na(v)1.5 in the heart, and Na(v)1.6 in the peripheral nervous system, respectively. In rainbow trout (Oncorhynchus mykiss), omNa(v)1.4a is the dominant isoform in its heart, accounting for over 80% of the Na(v) transcripts, while omNa(v)1.5a accounts for approximately 18%, and omNa(v)1.6a accounts for only 0.1%. Both omNa(v)1.4a and omNa(v)1.6a contain the aromatic amino acids phenylalanine and tyrosine, respectively, at the critical position 401 of the TTX-binding site in the I domain, conferring them high sensitivity to TTX. Even more surprisingly, omNa(v)1.5a also contains an aromatic tyrosine at this position, instead of the cysteine found in the mammalian TTX-resistant Na(v)1.5. Conclusion: The ortholog of mammalian skeletal muscle isoform omNa(v)1.4a is the major Na(v)α subunit in trout heart. All trout heart isoforms contain an aromatic residue at position 401, making fish heart I(Na) highly sensitive to TTX. TTX inhibits voltage-gated sodium channels in a highly efficient and selective manner without affecting any other receptor and ion channel systems. Tetrodotoxin (TTX) blocks sodium channels only from the outside of the nerve membrane; its mechanism of action is by binding to selective filters to prevent sodium ion flow. It does not impair the channel gating mechanism. In recent years, sodium channels tolerant to TTX have been discovered in the nervous system and have attracted much attention due to their role in pain perception. It is now known that TTX is not produced by inhalers but by bacteria and spreads to various animals through the food chain. Therapeutic Uses: Corneal damage can lead to photophobia, i.e., an aversion to light. We evaluated whether increased photophobia induced by corneal injury in rats was due to enhanced corneal afferent nerve activity through topical application of the local anesthetic lidocaine and the selective voltage-gated sodium channel blocker tetrodotoxin (TTX). Twenty-four hours after corneal ablation-induced corneal injury, exposure to strong light (460–485 nm) for 30 seconds induced an enhanced eye-closing response. While topical lidocaine did not affect the baseline eye-closing response to strong light in control rats, it eliminated the enhanced photophobia following corneal injury. Similarly, topical TTX did not affect the eye-closing response to strong light in rats with intact corneas but significantly reduced photophobia in rats with corneal injury. Given the known corneal toxicity of local anesthetics, we recommend TTX as a treatment option for photophobia and other clinical symptoms that may be associated with corneal nociceptor sensitization. Outlook: We found that both lidocaine and TTX can alleviate photophobia induced by corneal injury. Although corneal toxicity limits the use of local anesthetics, TTX may be a safer treatment option to alleviate photophobia associated with corneal injury. PMID: 26086898 Full text: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4664153 Green PG et al.; J Pain 16 (9): 881-6 (2015)
/EXPL THER/ Burns are considered the leading cause of injury in 5% of U.S. service members withdrawn from Operation Iraqi Freedom and Operation Enduring Freedom. Severe burn-related pain is often treated with opioids such as fentanyl, morphine, and methadone. Side effects of opioids include respiratory depression, cardiac depression, decreased motor and cognitive function, and the development of hyperalgesia, tolerance, and dependence. These effects have prompted the search for novel analgesics to treat burn-related pain in wounded combatants. Tetrodotoxin (TTX) is a selective voltage-gated sodium channel blocker and is currently undergoing clinical trials as an analgesic. A Phase III clinical trial for cancer-related pain has been completed, and a Phase III clinical trial for chemotherapy-induced neuropathic pain is planned. In mouse studies, TTX has also been shown to inhibit the development of chemotherapy-induced neuropathic pain. TTX, originally a neurotoxin discovered in marine animals, has now been shown to be safe for humans at therapeutic doses. The analgesic effect of TTX is believed to be achieved by inhibiting the influx of Na(+) ions required to initiate and conduct nociceptive impulses. A TTX-sensitive sodium channel, Nav1.7, has been shown to be crucial in reducing the thermal pain threshold after burns. To date, the analgesic effect of TTX has not been tested in burn-related pain. Male Sprague-Dawley rats suffered full-thickness burns to their right hind paw. TTX (8 μg/kg) was administered subcutaneously once daily, starting on day 3 post-burn and continuing until day 7 post-burn. Thermal hyperalgesia and mechanodynia were assessed at 60 and 120 minutes after each TTX treatment. TTX significantly reduced thermal hyperalgesia at all test time points, with a weaker but still statistically significant inhibitory effect on mechanical analgesia. These results suggest that systemic TTX may be an effective and rapidly acting battlefield burn analgesic, and could potentially replace or reduce the need for opioid analgesics. PMID:26424077 Salas MM et al.; Neuroscience Letters 607: 108-113 (2015)
/Exploring Treatments/ Persistent muscle pain is a common and disabling symptom with limited efficacy of existing treatments. Given the significant analgesic effect of tetrodotoxin (TTX) in a persistent skin pain model, we tested its local analgesic effect in a rat model of muscle pain induced by inflammation, ergonomic injury, and chemotherapy-induced neuropathy. Although local injection of TTX (0.03–1 μg) into the gastrocnemius muscle did not affect the mechanoresonance threshold in untreated rats, exposure to the inflammagenic carrageenan resulted in significant muscle mechanoresonance, which TTX dose-dependently inhibited. This anti-hyperalsonance effect remained significant after 24 hours. TTX also demonstrated significant analgesic effects on mechanoresonance induced by eccentric movement of the gastrocnemius muscle (an ergonomic pain model). Furthermore, TTX produced a small but significant inhibitory effect on neuromuscular pain induced by systemic injection of the anticancer chemotherapy drug oxaliplatin. These results suggest that TTX-sensitive sodium currents in nociceptors play a central role in various states of skeletal muscle nociceptive sensitization, supporting the idea that TTX-based therapeutic interventions may be effective for treating muscle pain. PMID:26548414 Full text: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4679288 Alvarez P, Levine JD; Neuroscience 311: 499-507 (2015)
/Exploring Treatment/ Objective: This study evaluated the efficacy of subcutaneous tetrodotoxin (TTX) for moderate to severe, poorly controlled cancer-related pain. Methods: Eligible patients were randomly assigned to receive TTX (30 μg) or placebo, administered subcutaneously twice daily for four consecutive days. Efficacy was assessed using pain and composite endpoints (including pain and quality of life indicators), and safety was assessed using standard methods. Results: A total of 165 patients were recruited from 19 research centers in Canada, Australia, and New Zealand, of whom 149 were included in the “intent-to-treat” population of the primary analysis. The primary analysis results support a clinical benefit of TTX compared to placebo, with a clinically significant effect size estimate of 16.2% (p = 0.0460) based solely on the pain endpoint. After pre-specified (Bonferroni-Holm) adjustment for both primary endpoints, the p-values were nominally statistically significant but not significant at the pre-specified two-sided 5% level. The mean duration of the analgesic response was 56.7 days (TTX group) and 9.9 days (placebo group). The most common adverse events were nausea, dizziness, and numbness or tingling in the mouth, which were generally mild to moderate and transient. Conclusion: Despite the small sample size, this study demonstrates a clinically meaningful analgesic signal. TTX may provide clinically meaningful analgesia for patients with persistent moderate to severe cancer pain despite optimal analgesia. PMID:28555092

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Recent reports indicate that sodium channel blockers can act as protective agents against hypoxia-induced neuronal damage, including protecting against anaerobic glycolysis. Therefore, this study investigated the effects of tetrodotoxin (TTX) and (±)-carvaline on brain vesicles in hypoxic rats, specifically lactate synthesis, vesicle ATP content, and cytoplasmic free sodium ion ([Na+]i) and calcium ion ([Ca2+]i) concentrations. Cytoplasmic free sodium ion and calcium ion concentrations were measured using SBFI and FURA-2 fluorescence methods, respectively. After hypoxia, basal lactate production increased from 2.9 nmol lactate/min/mg protein to 9.8 nmol lactate/min/mg protein. Although lactate synthesis appeared to remain stable for at least 45 minutes under hypoxia (inferred from the linear change in lactate production), ATP content continued to decline with a half-life (τ) of 14.5 minutes, indicating that anaerobic glycolysis was insufficient to meet the energy requirements of hypoxic vesicles. Correspondingly, after 6.3 minutes of hypoxia, [Na+]i and [Ca2+]i continued to increase, rising by 22.1 mmol/L Na+ and 274.9 nmol/L Ca2+, respectively. Further stimulation of the vesicles with veratrine accelerated the rate of ATP decline (τ = 5.1 min) and triggered a significant Na+ overload, with the Na+ concentration stabilizing at 119 mmol/L within minutes. Simultaneously, [Ca2+]i increased linearly at a rate of 355 nmol Ca2+/L/min. Despite the severe disturbance of ion homeostasis, lactate production was unaffected during the first 8 minutes of veratrine stimulation. However, after 30 minutes of veratrine addition, lactate synthesis was completely inhibited. If sodium channel blockers TTX and (±)-carvaline are used before hypoxia, vesicle ATP levels can be maintained, hypoxia-induced increases in [Na+]i and [Ca2+]i can be reduced, and veratrine-induced increases in [Na+]i and [Ca2+]i and inhibition of lactate production can be prevented. Data show that during hypoxia, voltage-dependent sodium channel-mediated massive sodium influx accelerates the decrease in ATP and triggers an increase in [Ca2+]i. Veratrine-induced massive sodium and calcium overload does not directly affect lactate synthesis, but it initiates the inhibition of lactate synthesis. © 1997 Elsevier Science Ltd. All rights reserved. [1]

C11H17N3O8

319.27

319.101

C, 41.38; H, 5.37; N, 13.16; O, 40.09

4368-28-9

Citrate-buffered Tetrodotoxin (TTX); 4368-28-9

11174599

Crystals

2.8±0.1 g/cm3

702.6±70.0 °C at 760 mmHg

225ºC dec

378.7±35.7 °C

0.0±5.0 mmHg at 25°C

2.087

2.16

8

9

1

22

562

9

C([C@@]1([C@@H]2[C@@H]3[C@H](NC(=N)N[C@]34[C@@H]([C@H]1O[C@@]([C@H]4O)(O)O2)O)O)O)O

CFMYXEVWODSLAX-QOZOJKKESA-N

InChI=1S/C11H17N3O8/c12-8-13-6(17)2-4-9(19,1-15)5-3(16)10(2,14-8)7(18)11(20,21-4)22-5/h2-7,15-20H,1H2,(H3,12,13,14)/t2-,3-,4-,5+,6-,7+,9+,10-,11+/m1/s1

(1R,5R,6R,7R,9S,11S,12S,13S,14S)-3-amino-14-(hydroxymethyl)-8,10-dioxa-2,4-diazatetracyclo[7.3.1.17,11.01,6]tetradec-3-ene-5,9,12,13,14-pentol

TETRODOTOXIN; Spheroidine; Tarichatoxin; Fugu poison; Maculotoxin; TTX; Tetrodotoxine; 4368-28-9; Babylonia japonica toxin 1; Tetrodoxin; Tectin;

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)

H2O: ~1 mg/mL
Stable Solution: Prepare 1 mg/mL in a dilute citrate or acetate buffer (pH 4-5).
Storage: Aqueous solutions at pH 4-5 are stable when frozen.
Instability: The compound is unstable in strong acid or alkaline solutions and is destroyed by boiling at pH 2.

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.)