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
Twenty-three specimens of a tree-frog Polypedates sp. were collected from two locations (Mymensingh and Barisal) of Bangladesh in 1999, and assayed for their toxicity scores and toxin principle. Among the tissues, only the skin of the Mymensingh specimens was found to be toxic in mouse test, with the toxicity scores of 31-923 ug/g. The toxin isolated from the skin was analyzed by high-performance liquid chromatography, electrospray ionization-time of flight mass spectrometry and proton nuclear magnetic resonance, and characterized as tetrodotoxin, a toxin principle.
Tetrodotoxin (TTX) and its analogs (TTXs), widely distributed among marine as well as terrestrial animals, induce dangerous intoxications. These highly potential toxins are also known as the causative agent of puffer fish poisoning. ... TTX, anhydrotetrodotoxin, 11-deoxytetrodotoxin and trideoxytetrodotoxin were determined in separated tissues of Bangladeshi marine puffers, Takifugu oblongus. TTX was predominant in skin, muscle and liver, whereas trideoxytetrodotoxin preponderated in the ovary. The toxicity of the various tissues was determined by a mouse bioassay.
To investigate the relationship between the toxicity of puffer fish and the distribution of tetrodotoxin-producing bacteria in puffer fish Fugu rubripes collected from the Bohai Sea of China, bacteria were isolated from each organ (ovaries, livers, intestines and gallbladders) and screened for tetrodotoxin (TTX) production. 20 out of 36 isolated strains were found to produce TTX in vitro. In the organs of ovaries and livers whose toxicity is more potent than other organs, the number and toxicity of TTX-producing strains was greater than that of others. Most TTX-producing bacterial strains were identified as Bacillus spp. (19 strains) and Actinomycete spp. (1 strain) based on the morphological observation, physiological and biochemical characteristics and G+C content of DNA. The purified toxin was identified to be TTX by high performance liquid chromatography assay, thin-layer chromatography assay and electrospray ionization mass spectrometry analysis. Our results suggested that TTX-producing bacteria are closely related to the toxification of the puffer fish. More research is needed to elucidate the mechanism of TTX synthesis and the role of TTX in bacteria.
The liver homogenate of puffer fish was fractionated into blood cell, nuclear, mitochondrial, microsomal and cytosol fractions by the differential centrifugation method. ... Analyses by HPLC and LC-FABMS demonstrated that tetrodotoxin is the major toxic principle in each fraction. These results reveal that tetrodotoxin is widely distributed in organelles in liver cells, though predominantly in the cytosol fraction.
For more Absorption, Distribution and Excretion (Complete) data for Tetrodotoxin (9 total), please visit the HSDB record page.

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Metabolism / Metabolites
The metabolic source of tetrodotoxin is uncertain. No algal source has been identified, and until recently tetrodotoxin was assumed to be a metabolic product of the host. However, recent reports of the production of tetrodotoxin/anhydrotetrodotoxin by several bacterial species, including strains of the family Vibrionaceae, Pseudomonas sp., and Photobacterium phosphoreum, point toward a bacterial origin of this family of toxins.
To investigate the genes related to the biosynthesis or accumulation of tetrodotoxin (TTX) in pufferfish, mRNA expression patterns in the liver from pufferfish, akamefugu Takifugu chrysops and kusafugu Takifugu niphobles, were compared by mRNA arbitrarily primed reverse transcription-polymerase chain reaction (RAP RT-PCR) with fish bearing different concentrations of TTX and its derivatives. RAP RT-PCR provided a 383 bp cDNA fragment and its transcripts were higher in toxic than non-toxic pufferfish liver. Its deduced amino acid sequence was similar to those of fibrinogen-like proteins reported for other vertebrates. Northern blot analysis and rapid amplification of cDNA ends (RACE) revealed that the cDNA fragment of 383 bp was composed of at least three fibrinogen-like protein (flp) genes, flp-1, flp-2 and flp-3. Relative mRNA levels of flp-1, flp-2 and flp-3 showed a linear correlation with toxicity of the liver for two pufferfish species.

Toxicity Summary
IDENTIFICATION AND USE: Tetrodotoxin (TTX) is a solid. TTX contained in puffer, has become an extremely popular chemical tool in the physiological and pharmacological laboratories since discovery of its channel blocking action in the early 1960s. More recently, the TTX-resistant sodium channels have been discovered in the nervous system and received much attention because of their role in pain sensation. TTX is now known to be produced not by puffer but by bacteria, and reaches various species of animals via food chain. HUMAN STUDIES: TTX is a deadly neurotoxin which selectively inhibits Na(+) activation mechanism of nerve impulse, without affecting the permeability of K(+) ions. TTX interferes with the transmission of signals from nerves to muscles by blocking sodium channels. This results in rapid weakening and paralysis of muscles, including those of the respiratory tract, which can lead to respiratory arrest and death. TTX poisoning may either have rapid onset (10 to 45 minutes) or delayed onset (generally within 3 to 6 hours but rarely longer). Death may occur as early as 20 minutes, or as late as 24 hours, after exposure; but it usually occurs within the first 4 to 8 hours. Patient/victims who live through the acute intoxication in the first 24 hours usually recover without residual deficits. Symptoms may last for several days and recovery takes days to occur. Upon ingestion, at first stage TTX producing numbness and sensation of prickling and tingling (paresthesia) of the lips and tongue, followed by facial and extremity paresthesias and numbness, headache, sensations of lightness or floating, profuse sweating (diaphoresis), dizziness, salivation (ptyalism), nausea, vomiting (emesis), diarrhea, abdominal (epigastric) pain, difficulty moving (motor dysfunction), weakness (malaise), and speech difficulties. At the second stage there is increasing paralysis, first in the extremities, then in the rest of the body, and finally in the respiratory muscles; difficulty breathing or shortness of breath (dyspnea); abnormal heart rhythms (cardiac dysrhythmias or arrhythmia); abnormally low blood pressure (hypotension); fixed and dilated pupils (mydriasis); coma; seizures; respiratory arrest; and death. A number of case reports describe food poisoning with TTX. Most of these poisoning episodes occur from home preparation and consumption and not from commercial sources of the pufferfish. TTX was shown to lack genotoxic activity in vitro in human lymphocytes with or without metabolic activation. ANIMAL STUDIES: The clinical symptoms and signs of TTX poisoning in dogs treated with TTX by iv infusion were similar to those of anticholinesterase poisoning. TTX was found to be about fifty times less toxic and to have more delayed death occurrence to mice via oral route than that via i.p. injection. In rats brain serotonin level was significantly increased and reached its peak level after 4 hours of TTX administration. Brain acetylcholine, histamine, and norepinephrine levels were also significantly increased but reached peak level after 6 hours. The effect of the gonad extract from pufferfish was more significantly profound and of longer duration than the skin extract. On the other hand, brain epinephrine did not show any significant change during the experimental period. TTX administered iv in male rabbits at sublethal levels produced shock due to perfusion failure with lactacidemia, hypoproteinemia, increased bleeding time, decreased red cell mass, and decreased platelet count. The severity of poisoning was proportional to the magnitude of tetrodotoxin given. Hemorrhages in brain, liver, lung, and diaphragm were observed in necropsy study. Significant differences in susceptibility to TTX were found among five mouse strains tested. TTX was clearly shown to lack in vitro or in vivo genotoxic activity. ECOTOXICITY STUDIES: TTX and its analogs (TTXs), widely distributed among marine as well as terrestrial animals, induce dangerous intoxications. Besides helping to deter predators, TTX resistance enables pufferfishes to selectively feed on TTX-bearing organisms. However TTXs do not protect flatworms from Guam from their predators but instead are used to capture mobile prey.

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Interactions
The ability of a tetrodotoxin (TTX)-specific monoclonal antibody to confer passive protection against lethal TTX challenge was investigated. The monoclonal antibody, T20G10, has an estimated affinity for TTX of approximately 10-9 M and is about 50-fold less reactive with anhydrotetrodotoxin and unreactive with tetrodonic acid by competitive immunoassay. T20G10 specifically inhibited TTX binding in an in vitro radioligand receptor binding assay, but had no effect on the binding of saxitoxin to the sodium channel on rat brain membranes. In prophylaxis studies, mice were administered T20G10 via the tail vein 30 min prior to i.p. TTX challenge (10 ug/kg). Under these conditions, 100 micrograms T20G10 protected 6/6 mice, whereas 3/6 mice were protected with 50 micrograms T20G10. Non-specific control monoclonal antibody did not protect against lethality. Therapy studies simulating oral intoxication were performed with mice given a lethal dose of TTX by gavage in a suspension of non-fat dry milk in phosphate-buffered saline. Death occurred within 25-35 min in 6/6 mice not treated with T20G10. However, 500 ug T20G10 administered via the tail vein 10-15 min after oral TTX exposure prevented death in 6/6 mice. Lower doses of mAb conferred less protection. PMID:8585093

At 24 hours after coronary artery occlusion in dogs, lidocaine (4 mg/kg, iv) and tetrodotoxin (2 ug/kg, iv) showed marked antiarrhythmic activity. At 2-fold lower doses, neither substance alone had an effect on arrhythmias, but when administered together, they induced almost complete restoration of cardiac rhythm.

Batrachotoxin increased sodium uptake by synaptosomes. Veratridine also increased the sodium uptake. Tetrodotoxin blocked the effects of the above toxins.

Denervated muscle of mice was excised after 5-6 days and incubated in 0.5% papain at 28-9 °C for 5-8 minutes. The proteolytic treatment abolished the shielding of sodium ion channel by labile surface proteins by partial removal which restored the sensitivity of receptors to tetrodotoxin after impairment by denervation.

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Non-Human Toxicity Values
LD50 Mouse oral 0.435 mg/kg Lewis, R.J. Sr. (ed) Sax's Dangerous Properties of Industrial Materials. 11th Edition. Wiley-Interscience, Wiley & Sons, Inc. Hoboken, NJ. 2004., p. 1827

LD50 Mouse ip 0.008 mg/kg Lewis, R.J. Sr. (ed) Sax's Dangerous Properties of Industrial Materials. 11th Edition. Wiley-Interscience, Wiley & Sons, Inc. Hoboken, NJ. 2004., p. 1827

LD50 Mouse sc 0.008 mg/kg Lewis, R.J. Sr. (ed) Sax's Dangerous Properties of Industrial Materials. 11th Edition. Wiley-Interscience, Wiley & Sons, Inc. Hoboken, NJ. 2004., p. 1827

LD50 Mouse iv 0.009 mg/kg Lewis, R.J. Sr. (ed) Sax's Dangerous Properties of Industrial Materials. 11th Edition. Wiley-Interscience, Wiley & Sons, Inc. Hoboken, NJ. 2004., p. 1827

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.

An aminoperhydroquinazoline poison found mainly in the liver and ovaries of fishes in the order tetraodontiformes, which are eaten. The toxin causes paresthesia and paralysis through interference with neuromuscular conduction. Tetrodotoxin is being investigated by Wex Pharmaceuticals for the treatment of chronic and breakthrough pain in advanced cancer patients as well as for the treatment of opioid dependence.
Spheroidine has been reported in Takifugu flavidus, Takifugu rubripes, and other organisms with data available.
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. Although found in various species of fish (such as the pufferfish), newts, frogs, flatworms, and crabs, tetrodotoxin, for which there is no known antidote, is actually produced by bacteria such as Vibrio alginolyticus, Pseudoalteromonas tetraodonis, and other vibrio and pseudomonas bacterial species.
An aminoperhydroquinazoline poison found mainly in the liver and ovaries of fishes in the order TETRAODONTIFORMES, which are eaten. The toxin causes paresthesia and paralysis through interference with neuromuscular conduction.

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Drug Indication
For the treatment of chronic and breakthrough pain in advanced cancer patients as well as for the treatment of opioid dependence.

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Mechanism of Action
Tetrodotoxin binds to site 1 of the fast voltage-gated sodium channel located at the extracellular pore opening. The binding of any molecules to this site will temporarily disable the function of the ion channel. Saxitoxin and several of the conotoxins also bind the same site.
Sodium current (I(Na)) of the mammalian heart is resistant to tetrodotoxin (TTX) due to low TTX affinity of the cardiac sodium channel (Na(v)) isoform Na(v)1.5. To test applicability of this finding to other vertebrates, TTX sensitivity of the fish cardiac I(Na) and its molecular identity were examined. METHODS: Molecular cloning and whole-cell patch-clamp were used to examine alpha-subunit composition and TTX inhibition of the rainbow trout (Oncorhynchus mykiss) cardiac Na(v) respectively. ...: I(Na) of the trout heart is about 1000 times more sensitive to TTX (IC50 = 1.8-2 nm) than the mammalian cardiac I(Na) and it is produced by three Na(v)alpha-subunits which are orthologs to mammalian skeletal muscle Na(v)1.4, cardiac Na(v)1.5 and peripheral nervous system Na(v)1.6 isoforms respectively. Oncorhynchus mykiss (om) omNa(v)1.4a is the predominant isoform of the trout heart accounting for over 80% of the Na(v) transcripts, while omNa(v)1.5a forms about 18% and omNa(v)1.6a only 0.1% of the transcripts. OmNa(v)1.4a and omNa(v)1.6a have aromatic amino acids, phenylalanine and tyrosine, respectively, in the critical position 401 of the TTX binding site of the domain I, which confers their high TTX sensitivity. More surprisingly, omNa(v)1.5a also has an aromatic tyrosine in this position, instead of the cysteine of the mammalian TTX-resistant Na(v)1.5. CONCLUSIONS: The ortholog of the mammalian skeletal muscle isoform, omNa(v)1.4a, is the predominant Na(v)alpha-subunit in the trout heart, and all trout cardiac isoforms have an aromatic residue in position 401 rendering the fish cardiac I(Na) highly sensitive to TTX.
... TTX inhibits voltage-gated sodium channels in a highly potent and selective manner without effects on any other receptor and ion channel systems. TTX blocks the sodium channel only from outside of the nerve membrane, and is due to binding to the selectivity filter resulting in prevention of sodium ion flow. It does not impair the channel gating mechanism. More recently, the TTX-resistant sodium channels have been discovered in the nervous system and received much attention because of their role in pain sensation. TTX is now known to be produced not by puffer but by bacteria, and reaches various species of animals via food chain.

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Therapeutic Uses
/EXPL THER/ Corneal injury can produce photophobia, an aversive sensitivity to light. Using topical application of lidocaine, a local anesthetic, and tetrodotoxin (TTX), a selective voltage-sensitive sodium channel blocker, we assessed whether enhanced aversiveness to light induced by corneal injury in rats was caused by enhanced activity in corneal afferents. Eye closure induced by 30 seconds of exposure to bright light (460-485 nm) was increased 24 hours after corneal injury induced by de-epithelialization. Although the topical application of lidocaine did not affect the baseline eye closure response to bright light in control rats, it eliminated the enhancement of the response to the light stimulus after corneal injury (photophobia). Similarly, topical application of TTX had no effect on the eye closure response to bright light in rats with intact corneas, but it markedly attenuated photophobia in rats with corneal injury. Given the well-established corneal toxicity of local anesthetics, we suggest TTX as a therapeutic option to treat photophobia and possibly other symptoms that occur in clinical diseases that involve corneal nociceptor sensitization. PERSPECTIVE: We show that lidocaine and TTX attenuate photophobia induced by corneal injury. Although corneal toxicity limits use of local anesthetics, TTX may be a safer therapeutic option to reduce the symptom of 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/ Burn injuries have been identified as the primary cause of injury in 5% of U.S. military personnel evacuated from Operations Iraqi Freedom and Enduring Freedom. Severe burn-associated pain is typically treated with opioids such as fentanyl, morphine, and methadone. Side effects of opioids include respiratory depression, cardiac depression, decrease in motor and cognitive function, as well as the development of hyperalgesia, tolerance and dependence. These effects have led us to search for novel analgesics for the treatment of burn-associated pain in wounded combat service members. Tetrodotoxin (TTX) is a selective voltage-gated sodium channel blocker currently in clinical trials as an analgesic. A phase 3 clinical trial for cancer-related pain has been completed and phase 3 clinical trials on chemotherapy-induced neuropathic pain are planned. It has also been shown in mice to inhibit the development of chemotherapy-induced neuropathic pain. TTX was originally identified as a neurotoxin in marine animals but has now been shown to be safe in humans at therapeutic doses. The antinociceptive effects of TTX are thought to be due to inhibition of Na(+) ion influx required for initiation and conduction of nociceptive impulses. One TTX sensitive sodium channel, Nav1.7, has been shown to be essential in lowering the heat pain threshold after burn injuries. To date, the analgesic effect of TTX has not been tested in burn-associated pain. Male Sprague-Dawley rats were subjected to a full thickness thermal injury on the right hind paw. TTX (8 ug/kg) was administered once a day systemically by subcutaneous injection beginning 3 days post thermal injury and continued through 7 days post thermal injury. Thermal hyperalgesia and mechanical allodynia were assessed 60 and 120 min post injection on each day of TTX treatment. TTX significantly reduced thermal hyperalgesia at all days tested and had a less robust, but statistically significant suppressive effect on mechanical allodynia. These results suggest that systemic TTX may be an effective, rapidly acting analgesic for battlefield burn injuries and has the potential for replacing or reducing the need for opioid analgesics. PMID:26424077 Salas MM et al; Neurosci Lett 607: 108-113 (2015)

/EXPL THER/ Persistent muscle pain is a common and disabling symptom for which available treatments have limited efficacy. Since tetrodotoxin (TTX) displays a marked antinociceptive effect in models of persistent cutaneous pain, we tested its local antinociceptive effect in rat models of muscle pain induced by inflammation, ergonomic injury and chemotherapy-induced neuropathy. While local injection of TTX (0.03-1 ug) into the gastrocnemius muscle did not affect the mechanical nociceptive threshold in naive rats, exposure to the inflammogen carrageenan produced a marked muscle mechanical hyperalgesia, which was dose-dependently inhibited by TTX. This antihyperalgesic effect was still significant at 24 hr. TTX also displayed a robust antinociceptive effect on eccentric exercise-induced mechanical hyperalgesia in the gastrocnemius muscle, a model of ergonomic pain. Finally, TTX produced a small but significant inhibition of neuropathic muscle pain induced by systemic administration of the cancer chemotherapeutic agent oxaliplatin. These results indicate that TTX-sensitive sodium currents in nociceptors play a central role in diverse states of skeletal muscle nociceptive sensitization, supporting the suggestion that therapeutic interventions based on TTX may prove useful in the treatment of muscle pain. PMID:26548414 Full text: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4679288 Alvarez P, Levine JD; Neuroscience 311: 499-507 (2015)

/EXPL THER/ OBJECTIVE: This study evaluated subcutaneous injections of tetrodotoxin (TTX) for the treatment of moderate to severe, inadequately controlled cancer-related pain. METHODS: Eligible patients were randomized to receive TTX (30 ug) or placebo subcutaneously twice daily for four consecutive days. Efficacy was assessed using pain and composite endpoints (including pain and quality of life measures), and safety was evaluated using standard measures. RESULTS: 165 patients were enrolled at 19 sites in Canada, Australia, and New Zealand, with 149 patients in the primary analysis "intent-to-treat" population. The primary analysis supports a clinical benefit of TTX over placebo based on the pain endpoint alone with a clinically significant estimated effect size of 16.2% (p = 0.0460). The p value was nominally statistically significant after prespecified (Bonferroni Holm) adjustment for the two primary endpoints but not at the prespecified two-sided 5% level. The mean duration of analgesic response was 56.7 days (TTX) and 9.9 days (placebo). Most common adverse events were nausea, dizziness, and oral numbness or tingling and were generally mild to moderate and transient. CONCLUSIONS: Although underpowered, this study demonstrates a clinically important analgesic signal. TTX may provide clinically meaningful analgesia for patients who have persistent moderate to severe cancer pain despite best analgesic care. PMID:28555092

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Because recent reports point to Na+ channel blockers as protective agents directed against anoxia-induced neuronal damage including protection of anaerobic glycolysis, the influences of tetrodotoxin (TTX) and (±)-kavain on anoxic rat brain vesicles were investigated with respect to lactate synthesis, vesicular ATP content and cytosolic free Na+ and Ca2+ ([Na+]i, [Ca2+]i), both of the latter determined fluorometrically employing SBFI and FURA-2, respectively. After anoxia, basal lactate production was increased from 2.9 to 9.8 nmol lactate/min/mg protein. Although lactate synthesis seemed to be stable for at least 45 min of anoxia, as deduced from the linearity of lactate production, the ATP content declined continuously with a half life (τ) af 14.5 min, indicating that anaerobic glycolysis was insufficient to cover the energy demand of anoxic vesicles. Correspondingly, [Na+]i and [Ca2+]i increased persistently after anoxia by 22.1 mmol/l Na+ and 274.9 nmol/l Ca2+, determined 6.3 min after onset. An additional stimulation of vesicles with veratridine accelerated the drop of ATP (τ = 5.1 min) and provoked a massive Na+ overload, which levelled off to 119 mmol/l Na+ within a few minutes. Concomitantly, [Ca2+]i increased linearly with a rate of 355 nmol Ca2+/l/min. Despite the massive perturbation of ion homeostasis, lactate production was unaffected during the first 8 min of veratridine stimulation. However, complete inhibition of lactate synthesis took place 30 min after veratridine was added. The Na+ channel blockers TTX and (±)-kavain, if applied before anoxia, preserved vesicular ATP content, diminished anoxia-induced increases in [Na+]i and [Ca2+]i and prevented both the veratridine-induced increases of [Na+]i and [Ca2+]i and the inhibition of lactate production. The data indicate a considerable Na+ influx via voltage-dependent Na+ channels during anoxia, which speeds up the decline in ATP and provokes an increase in [Ca2+]i. A massive Na+ and Ca2+ overload induced by veratridine failed to influence lactate synthesis directly, but initiated its inhibition. © 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

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; 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)

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.)