Gonyautoxins 2/3 Local Periarticular Injection for Pain Management after Total Knee Arthroplasty: A Double-Blind, Randomized Study.

The purpose of this study was to compare the efficacy of periarticular infiltration of gonyautoxin 2/3 (GTX 2/3) and a mixture of levobupivacaine, ketorolac, and epinephrine for pain management after total knee arthroplasty (TKA). Forty-eight patients were randomly allocated to receive periarticular infiltration of 40 µg GTX 2/3 (n = 24) diluted in 30 mL of sodium chloride 0.9% (study group) or a combination of 300 mg of levobupivacaine, 1 mg of epinephrine, and 60 mg ketorolac (n = 24) diluted in 150 mL of sodium chloride 0.9% (control group). Intraoperative anesthetic and surgical techniques were identical for both groups. Postoperatively, all patients received patient-controlled analgesia (morphine bolus of 1 mg; lockout interval of 8 minutes), acetaminophen, and ketoprofen for 72 hours. A blinded investigator recorded morphine consumption, which was the primary outcome. Also, the range of motion (ROM) and static and dynamic pain were assessed at 6, 12, 36, and 60 hours after surgery. The incidence of adverse events, time to readiness for discharge, and length of hospital stay were also recorded. The median of total cumulative morphine consumption was 16 mg (range, 0-62 mg) in the GTX 2/3 group and 9 mg (range, 0-54 mg) in control group, which did not reach statistical difference (median test, p = 0.40). Furthermore, static and dynamic pain scores were similar at all time intervals. GTX 2/3 was inferior in range of motion at 6 and 12 hours; nevertheless, we noted no difference after 36 hours. No differences between groups were found in terms of complications, side effects, or length of hospital stay. No significant differences were found between groups in terms of breakthrough morphine requirement. However, local anesthetic use resulted in an increased ROM in the first 12 hours. This prospective randomized clinical trial shows that GTX 2/3 is a safe and efficient drug for pain control after TKA; nevertheless, more studies using GTX 2/3 with larger populations are needed to confirm the safety profile and efficiency. This is level 1 therapeutic study, randomized, double-blind clinical trial.

Neosaxitoxin, a Paralytic Shellfish Poison phycotoxin, blocks pain and inflammation in equine osteoarthritis.

The Osteoarthritis is a chronic disease characterized by a progressive deterioration of the articular cartilage producing a strong inflammatory activity and chronic pain in patients. Horses also show osteoarthritis. Since the activation and progression of the disease are similar to that of human we developed a study model in horses. In this study, we test the effect of Neosaxitoxin, a phycotoxin from Paralytic Shellfish Poison, in the remediation of osteoarthritis equine clinical symptoms such as pain (showed in lameness) and inflammation quantifying the amounts of pro-inflammatory markers like cellular infiltration, TNF-alpha and nitric oxide in the synovial fluid obtained from the horse damaged joint. The outcomes show that Neosaxitoxin blocks pain for long lasting period (average 24.7 days). Furthermore, the amounts of pro-inflammatory markers were reduced and consequently an enhanced horse's well-being was obtained. Neosaxitoxin showed to be a candidate for establishing treatment protocols for OA, being safe and effective as a pain blocker in equine osteoarthritis.

Neosaxitoxin Inhibits the Expression of Inflammation Markers of the M1 Phenotype in Macrophages.

(1) Background: Neosaxitoxin (NeoSTX) has been used as a local anesthetic, but its anti-inflammatory effects have not been well defined. In the present study, we investigate the effects of NeoSTX on lipopolysaccharide (LPS)-activated macrophages. (2) Methods: Raw 264.7 and equine PBMC cells were incubated with or without 100 ng/mL LPS in the presence or absence of NeoSTX (1µM). The expression of inflammatory mediators was assessed: nitric oxide (NO) content using the Griess assay, TNF-α content using the ELISA assay, and mRNA of inducible nitric oxide synthase (iNOS), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) using a real-time polymerase chain reaction. (3) Results: NeoSTX (1 µM) significantly inhibited the release of NO, TNF-α, and expression of iNOS, IL-1ß, and TNF-α in LPS-activated macrophages of both species studied. Furthermore, our study shows that the LPS-induced release of inflammatory mediators was suppressed by NeoSTX. Additionally, NeoSTX deactivated polarized macrophages to M1 by LPS without compromising its polarization towards M2. (4) Conclusions: NeoSTX inhibits LPS-induced release of inflammatory mediators from macrophages, and these effects may be mediated by the blockade of voltage-gated sodium channels (VGSC).

In vivo blockade of ovarian sympathetic activity by Neosaxitoxin prevents polycystic ovary in rats.

A high sympathetic tone is observed in the development and maintenance of the polycystic ovary (PCO) phenotype in rats. Neosaxitoxin (NeoSTX) specifically blocks neuronal voltage-dependent Na+ channels, and we studied the capacity of NeoSTX administered into the ovary to block sympathetic nerves and PCO phenotype that is induced by estradiol valerate (EV). The toxin was administered with a minipump inserted into the bursal cavity using two protocols: (1) the same day as EV administration and (2) 30 days after EV to block the final step of cyst development and maintenance of the condition. We studied the estrous cycling activity, follicular morphology, steroid plasma levels, and norepinephrine concentration. NeoSTX administered together with EV decreased NA intraovarian levels that were induced by EV, increased the number of corpora lutea, decreased the number of follicular cyst found after EV administration, and decreased the previously increased testosterone plasma levels induced by the PCO phenotype. Estrous cycling activity also recovered. NeoSTX applied after 30 days of EV administration showed near recovery of ovary function, suggesting that there is a specific window in which follicular development could be protected from cystic development. In addition, plasma testosterone levels decreased while those of progesterone increased. Our data strongly suggest that chronic inhibition of sympathetic nerves by a locally applied long-lasting toxin is a new tool to manage the polycystic phenotype in the rat and could be applied to other mammals depending on sympathetic nerve activity.

Evaluation of Neosaxitoxin as a local anesthetic during piglet castration: A potential alternative for Lidocaine.

OBJETIVE: To evaluate Neosaxitoxin (NeoSTX) as a local anesthetic drug, for pain control during and after piglet castration. STUDY DESIGN: Prospective, randomized and double-blind study. ANIMALS: 24 commercial hybrids, males, 23-day-old piglets. METHODS: The piglets were randomized into two groups: a Lidocaine group and a NeoSTX group. One minute before castration, they were injected intra-scrotally with a single dose of Lidocaine (20 mg, in 1 mL) and NeoSTX (0.1 µg, in 1 mL), respectively. RESULTS: NeoSTX does not generate vasoconstriction or scrotal contraction, unlike Lidocaine, where a decrease in temperature and scrotal size is observed within 5 min after the procedure. After 24 h, wound inflammation, as measured by scrotal size, was lower in the NeoSTX group. No significant difference could be shown between the vocalizations and facial expressions of pain of both groups during the castration procedure. CONCLUSIONS: A single dose of NeoSTX is safe and effective for pain management during and after piglet castration. NeoSTX treated piglets were less affected by castration than those in the Lidocaine group, thus reducing piglet stress and enhancing the quality of piglet convalescence.

Manejo de una paciente embarazada con déficit congénito de factor VII leve / Management of a pregnant patient with mild factor VII congenital deficiency

RESUMEN No hay guías específicas para el manejo de pacientes embarazadas con la deficiencia de Factor VII; no hay una correlación entre el nivel de FVII y el riesgo de hemorragia y el nivel del Factor VII aumento durante el embarazo. Presentamos un caso clínico, el manejo y las recomendaciones del consenso.

Neosaxitoxin, a Paralytic Shellfish Poison toxin, effectively manages bucked shins pain, as a local long-acting pain blocker in an equine model.

Local anesthesia is an effective method to control pain. Neosaxitoxin is a phycotoxin whose molecular mechanism includes a reversible inhibition of voltage-gated sodium channels at the axonal level, impeding nerve impulse propagation. The present study was designed to evaluate the clinical efficacy of Neosaxitoxin as a local long-acting pain blocker in horse bucked shins, and it was found to effectively control pain. While Neosaxitoxin and Gonyautoxin, another Paralytic Shellfish Poison (PSP) toxin, have been successfully used in humans as long-lasting pain blockers, this finding marks the first time a PSP has been shown to have an established effect in veterinary medicine.

Sublethal doses of dinophysistoxin-1 and okadaic acid stimulate secretion of inflammatory factors on innate immune cells: Negative health consequences.

One of the proposed mechanisms to explain why Diarrhetic Shellfish Poison (DSP) toxins are tumor promoters is founded on the capacity of these toxins to increase TNF-α secretion. Although macrophages are the principal cells in the activation of the inflammatory response, the immune profile that Okadaic acid (OA) and Dinophysistoxin-1 (DTX-1) trigger in these cells has not been fully explored. We have therefore investigated the effect of various concentrations of both toxins on the activity of several inflammatory factors. Our results demonstrate that OA and DTX-1, at sublethal doses, stimulate secretion of inflammatory factors. Nevertheless DTX-1 was more potent than OA in increasing TNF-α and IL-6 as well as their dependent chemokines KC, MCP-1, LIX, MIP-1 α, MIP-1 ß and MIP-2. On the other hand, secretion of IFN-γ and the anti-inflammatory cytokines, IL-4 and IL-10, was unaffected. In addition, DTX-1 also raises matrix metalloproteinase-9 (MMP-9) activity. In this study, for the first time the effect of OA and DTX-1 over the secretion of pro-inflammatory and carcinogenic signals in macrophages are compared, showing that DTX-1 is ten times more potent that OA. The inflammatory profile produced by DTX-1 is shown for the first time. The safe limit regulation should be changed to DSP toxins zero tolerance in the shellfish to be consumed by humans.

Gonyautoxins: First evidence in pain management in total knee arthroplasty.

Improvements in pain management techniques in the last decade have had a major impact on the practice of total knee arthroplasty (TKA). Gonyautoxin are phycotoxins, whose molecular mechanism of action is a reversible block of the voltage-gated sodium channels at the axonal level, impeding nerve impulse propagation. This study was designed to evaluate the clinical efficacy of Gonyautoxin infiltration, as a long acting pain blocker in TKA. Fifteen patients received a total dose of 40 µg of Gonyautoxin during the TKA operation. Postoperatively, all patients were given a standard painkiller protocol: 100 mg of intravenous ketoprofen and 1000 mg of oral acetaminophen every 8 hours for 3 days. The Visual Analog Scale (VAS) pain score and range of motion were recorded 12, 36, and 60 hours post-surgery. All patients reported pain of 2 or less on the VAS 12 and 36 hours post-surgery. Moreover, all scored were less than 4 at 60 hours post-surgery. All patients achieved full knee extension at all times. No side effects or adverse reactions to Gonyautoxin were detected in the follow-up period. The median hospital stay was 3 days. For the first time, this study has shown the effect of blocking the neuronal transmission of pain by locally infiltrating Gonyautoxin during TKA. All patients successfully responded to the pain control. The Gonyautoxin infiltration was safe and effective, and patients experienced pain relief without the use of opioids.

First evidence of neosaxitoxin as a long-acting pain blocker in bladder pain syndrome.

INTRODUCTION AND HYPOTHESIS: Neosaxitoxin is a phycotoxin whose molecular mechanism of action shows a reversible inhibition of voltage-gated sodium channels at the axonal level, impeding nerve impulse propagation. This study was designed to evaluate the clinical efficacy of neosaxitoxin as a long-acting pain blocker in the treatment of bladder pain syndrome (BPS). METHODS: Five patients with a diagnosis of BPS received a total dose of 80 µg of neosaxitoxin in an isoosmotic solution of 0.9 % NaCl, pH 6.5. Infiltration was performed via cystoscopy under spinal anesthesia. Questionnaires were administered immediately before and 7, 30 and 90 days after the procedure to measure the patients' reported pain severity and quality of life. RESULTS: This study, for the first time, showed the effect of blocking the neuronal transmission of pain by local infiltration of neosaxitoxin into the bladder submucosa. All five patients successfully responded to the treatment. Furthermore, the analgesic effect lasted for the entire 90 days of follow-up without the need for a second infiltration, and no adverse reactions to neosaxitoxin were detected. CONCLUSIONS: Neosaxitoxin infiltration was shown to be a safe and effective intervention to control pain related to BPS. It was well tolerated by patients, who experienced extended pain relief and associated beneficial effects over a follow-up of 90 days. These results confirm the effectiveness of neosaxitoxin as a long-acting local pain blocker.

Management of Pregnancy in a Chilean Patient with Congenital Deficiency of Factor VII and Glanzmann's Thrombasthenia Variant.

Patients with inherited bleeding disorders are rare in obstetric practice but present with prolonged bleeding even after minor invasive procedures. They require a combined approach with obstetric and hematological management of each case, including the neonatal management of a possibly affected fetus. We present the case of a pregnancy in a patient with combined Factor VII deficiency and Glanzmann's thrombasthenia, the successful obstetric and hematological management of the case, and a review of the literature.

Okadaic acid toxin at sublethal dose produced cell proliferation in gastric and colon epithelial cell lines.

The aim of this study was to analyze the effect of Okadaic Acid (OA) on the proliferation of gastric and colon epithelial cells, the main target tissues of the toxin. We hypothesized that OA, at sublethal doses, activates multiple signaling pathways, such as Erk and Akt, through the inhibition of PP2A. To demonstrate this, we carried out curves of doses and time response against OA in AGS, MKN-45 and Caco 2 cell lines, and found an increase in the cell proliferation at sublethal doses, at 24 h or 48 h exposure. Indeed, cells can withstand high concentrations of the toxin at 4 h exposure, the time chosen considering the maximum time before total gastric emptying. We have proved that this increased proliferation is due to an overexpression of Cyclin B, a cyclin that promotes the passage from G2 to mitosis. In addition, we have demonstrated that OA induces activation of Akt and Erk in the three cells lines, showing that OA can activate pathways involved in oncogenesis. In conclusion, this study contributes to the knowledge about the possible effects of chronic OA consumption.

Determination of the variability of both hydrophilic and lipophilic toxins in endemic wild bivalves and carnivorous gastropods from the southern part of Chile.

The aim of this study was to analyse and determine the composition of paralytic shellfish poisoning (PSP) toxins and lipophilic toxins in the Region of Aysén, Chile, in wild endemic mussels (Mytilus chilensis, Venus antiqua, Aulacomya ater, Choromytilus chorus, Tagelus dombeii and Gari solida) and in two endemic carnivorous molluscs species (Concholepas concholepas and Argobuccinum ranelliforme). PSP-toxin contents were determined by using HPLC with fluorescence detection, while lipophilic toxins were determined by using LC-MS/MS. Mean concentrations for the total of PSP toxins were in the range 55-2505 µg saxitoxin-equivalent/100 g. The two most contaminated samples for PSP toxicity were bivalve Gari solida and carnivorous Argobuccinum ranelliforme with 2505 ± 101 and 1850 ± 137 µg saxitoxin-equivalent/100 g, respectively (p < 0.05). The lipophilic toxins identified were okadaic acid, dinophysistoxin-1 (DTX-1), azaspiracid-1 (AZA-1), pectenotoxin-2 (PTX-2) and yessotoxins (YTX). All analysed molluscs contained lipophilic toxins at levels ranging from 56 ± 4.8 to 156.1 ± 8.2 µg of okadaic acid-equivalent/kg shellfish together with YTX at levels ranging from 1.0 ± 0.1 to 18 ± 0.9 µg of YTX-equivalent/kg shellfish and AZA at levels ranging from 3.6 ± 0.2 to 31 ± 2.1 µg of AZA-equivalent/kg shellfish. Furthermore, different bivalves and gastropods differ in their capacity of retention of lipophilic toxins, as shown by the determination of their respective lipophilic toxins levels. In all the evaluated species, the presence of lipophilic toxins associated with biotransformation in molluscs and carnivorous gastropods was not identified, in contrast to the identification of PSP toxins, where the profiles identified in the different species are directly related to biotransformation processes. Thus, this study provides evidence that the concentration of toxins in the food intake of the evaluated species (Bivalvia and Gastropoda class) determines the degree of bioaccumulation and biotransformation they will thereafter exhibit.

Lipophilic toxin profiles detected in farmed and benthic mussels populations from the most relevant production zones in Southern Chile.

Lipophilic toxins associated with diarrhoeic toxins were found in Mytilus chilensis (Blue mussels) and Aulacomya ater (Ribbed mussels). These shellfish samples were collected from Chiloe Island, Southern Chile. The samples were tested by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After the analysis, four toxins were found: DTX-1, DTX-3, YTX and PTX. All toxins were identified by comparing their HPLC retention times with those of analytical standards and confirmed by LC-MS/MS. Dinophysistoxin-1 (DTX-1) and dinophysistoxin-3 (DTX-3) toxins were the major components within the mussel extracts. Nevertheless, the percentages of these toxins differed depending on the area they were collected from and/or the sampling date. The levels detected in Butacheuques Island for okadaic acid (OA) was 267 ± 3.5 µg OA eq kg(-1) (p < 0.05) and for DTX-3 was 183.4 ± 7.5 µg kg(-1) in ribbed mussels. Pectenotoxin (PTX) and yessotoxin (YTX) were the toxins detected in minor proportions in the toxic profile of the bivalves. The maximum concentration of YTX detected in ribbed mussels was 85.2 ± 2.8 µg kg(-1) in Mechuque Island, whereas the PTX-2 level in ribbed mussels was 82.0 ± 2.4 µg kg(-1) in Cailin Island. Analogues of YTX and PTX-2 were not detected in any of the analysed mussels, which did not support the supposed presence of isomers of toxins as a result of the enzymatic metabolism of bivalves. This study found evidence proving co-occurrence of lipophilic toxins - like PTX and YTX - with diarrhoeic toxin in samples collected in Southern Chile, which is, to date, the more complex mix of lipophilic toxins ever found in mussels samples from Southern Chile.

Highly toxic Microcystis aeruginosa strain, isolated from São Paulo-Brazil, produce hepatotoxins and paralytic shellfish poison neurotoxins.

While evaluating several laboratory-cultured cyanobacteria strains for the presence of paralytic shellfish poison neurotoxins, the hydrophilic extract of Microcystis aeruginosa strain SPC777--isolated from Billings's reservoir, São Paulo, Brazil--was found to exhibit lethal neurotoxic effect in mouse bioassay. The in vivo test showed symptoms that unambiguously were those produced by PSP. In order to identify the presence of neurotoxins, cells were lyophilized, and the extracts were analyzed by HPLC-FLD and HPLC-MS. HPLC-FLD analysis revealed four main Gonyautoxins: GTX4(47.6%), GTX2(29.5%), GTX1(21.9%), and GTX3(1.0%). HPLC-MS analysis, on other hand, confirmed both epimers, with positive Zwitterions M(+) 395.9 m/z for GTX3/GTX2 and M(+) 411 m/z for GTX4/GTX1 epimers.The hepatotoxins (Microcystins) were also evaluated by ELISA and HPLC-MS analyses. Positive immunoreaction was observed by ELISA assay. Alongside, the HPLC-MS analyses revealed the presence of [L: -ser(7)] MCYST-RR. The N-methyltransferase (NMT) domain of the microcystin synthetase gene mcyA was chosen as the target sequence to detect the presence of the mcy gene cluster. PCR amplification of the NMT domain, using the genomic DNA of the SPC777 strain and the MSF/MSR primer set, resulted in the expected 1,369 bp product. The phylogenetic analyses grouped the NMT sequence with the NMT sequences of other known Microcystis with high bootstrap support. The taxonomical position of M. aeruginosa SPC777 was confirmed by a detailed morphological description and a phylogenetic analysis of 16S rRNA gene sequence. Therefore, co-production of PSP neurotoxins and microcystins by an isolated M. aeruginosa strain is hereby reported for the first time.

First evidence of Okadaic acid acyl-derivative and Dinophysistoxin-3 in mussel samples collected in Chiloe Island, Southern Chile.

This paper shows the detection of Diarrhetic Shellfish Poison (DSP) phycotoxins, using HPLC-FLD with pre-column derivatization procedure and HPLC-MS methods, in the analysis of shellfish extracts tested positive with the official DSP mouse bioassay. The shellfish samples were collected in Chiloe Island, Southern of Chile. The amount of Dinophysistoxin-3 (DTX-3) measured in the shellfish extracts were in average above the international safe limits for DSP content in the shellfish extracts analyzed. As internal control of detection and recovery, DTX-1 analytical standard was spiked into dichloromethane-clean shellfish extracts in order to calculate de extraction recovery of DTX-1. The average recovery was 97%. From all DSP toxins analyzed, the hydrolyzed extract samples appeared mainly DTX-3 in concentrations ranging from 99.40 +/- 1.22 to 257.73 +/- 12.46 ng/g digestive-glands. The acyl-Okadaic Acid (acyl-OA) was also detected in some samples, ranging from 1.02 +/- 1.4 to 3.07. +/- 1.6 ng of DSP toxin/g digestive-glands. This is the first report of acyl-OA ever found in Chilean shellfish samples. This data shows that shellfish samples were contaminated with a complex DSP toxins profile, in which DTX-3 is the major DSP toxin component, followed by DTX-1 and the acyl-OA as the minor one. The important findings showed in this study are the presence of both acyl-derivates (DTX-3 and Acyl-OA) which are the product of a main metabolic biotransformation that occurred inside the shellfish, in order to chelate DTX-1 and OA, transforming them into DTX-3 and the acyl-OA respectively. This metabolic biotransformation must be performed to avoid self-inhibition of their Protein Phosphatase 2A done by DTX-1 and OA, since both acyl-derivates (DTX-3 and acyl-OA) do not inhibit Protein Phosphatase 2A. This complex DSP toxins profile and the permanent presence of both acyl-derivates (DTX-3 and Acyl-OA) could explain the permanent diarrhea symptoms that experience patients who have ingested cooked shellfish in the southern of Chile. This diarrhea is not associated to Vibrio parahaemolyticus or other enteropathogens as had been suggested before. The massive shellfish consumption is an important Chilean cultural habit and now has become a major health issue in the southern of Chile.

Route of metabolization and detoxication of paralytic shellfish toxins in humans.

Paralytic shellfish toxins (PST) are a collection of over 26 structurally related imidazoline guanidinium derivatives produced by marine dinoflagellates and freshwater cyanobacteria. Glucuronidation of drugs by UDP-glucuronosyltransferase (UGT) is the major phase II conjugation reaction in mammalian liver. In this study, using human liver microsomes, the in vitro paralytic shellfish toxins oxidation and sequential glucuronidation are achieved. Neosaxitoxin (neoSTX), Gonyautoxin 3/2 epimers (GTX3/GTX2) and Saxitoxin (STX) are used as starting enzymatic substrates. The enzymatic reaction final product metabolites are identified by using HPLC-FLD and HPLC/ESI-IT/MS. Four metabolites from GTX3/GTX2 epimers precursors, three of neoSTX and two of STX are clearly identified after incubating with UDPGA/NADPH and fresh liver microsomes. The glucuronic-Paralytic Shellfish Toxins were completely hydrolysed by treatment with beta-glucuronidase. All toxin analogs were identified comparing their HPLC retention time with those of analytical standard reference samples and further confirmed by HPLC/ESI-IT/MS. Paralytic Shellfish Toxins (PST) were widely metabolized by human microsomes and less than 15% of the original PST, incubated as substrate, stayed behind at the end of the incubation. The apparent V(max) and Km formation values for the respective glucuronides of neoSTX, GTX3/GTX2 epimers and STX were determined. The V(max) formation values for Glucuronic-GTX3 and Glucuronic-GTX2 were lower than Glucuronic-neoSTX and Glucuronic-STX (6.8+/-1.9x10(-3); 8.3+/-2.8x10(-3) and 9.7+/-2.8x10(-3)pmol/min/mg protein respectively). Km of the glucuronidation reaction for GTX3/GTX2 epimers was less than that of glucuronidation of neoSTX and STX (20.2+/-0.12; 27.06+/-0.23 and 32.02+/-0.64microM respectively). In conclusion, these data show for the first time, direct evidence for the sequential oxidation and glucuronidation of PST in vitro, both being the initial detoxication reactions for the excretion of these toxins in humans. The PST oxidation and glucuronidation pathway showed here, is the hepatic conversion of its properly glucuronic-PST synthesized, and the sequential route of PST detoxication in human.

Potentiation of local anesthetic activity of neosaxitoxin with bupivacaine or epinephrine: development of a long-acting pain blocker.

Local anesthetics effectively block and relieve pain, but with a relatively short duration of action, limiting its analgesic effectiveness. Therefore, a long-acting local anesthetic would improve the management of pain, but no such agent is yet available for clinical use. The aim of this study is to evaluate the potentiation of the anesthetic effect of neosaxitoxin, with bupivacaine or epinephrine in a randomized double-blind clinical trial. Ten healthy males were subcutaneously injected into the left and right forearms with a randomized pair of the following treatments: (i) bupivacaine (5 mg); (ii) neosaxitoxin (10 microg); (iii) neosaxitoxin (10 microg) plus bupivacaine (5 mg), and (iv) neosaxitoxin (10 microg) plus epinephrine (1:100.000), but all participant received all four formulations (in 2 ml; s.c.), with 1 month elapsing between the two round of experiments. A validated sensory and pain paradigm was used for evaluating the effect of the treatment 0-72 h after the injections, measuring sensory, pain, and mechanical touch perception threshold. The duration of the effect produced by combined treatments was longer than that by the single drugs. In conclusion, bupivacaine and epinephrine potentiate the local anesthetic effect of neosaxitoxin in humans when co-injected subcutaneously. The present results support the idea that neosaxitoxin is a new long-acting local pain blocker, with highly potential clinical use.

Evidence of in vitro glucuronidation and enzymatic transformation of paralytic shellfish toxins by healthy human liver microsomes fraction.

Paralytic Shellfish Toxins (PST) are endemic components found in filter bivalves in Southern Chile. Post-mortems analysis of fluid and tissue samples has shown biotransformation of PST in humans. The Gonyautoxin 3 (GTX3) and Gonyautoxin 2 (GTX2) are the major PST components in the toxin profile found in Chilean shellfish extracts, being as much as 65% of the total content of PST in filter bivalves. Therefore, they are the major accountable components of the human intoxication by shellfish consumption. The aim of this study is to show in vitro glucuronidation and biotransformation of GTX3 and GTX2 when they are incubated with microsomal fraction isolated from healthy human livers. Microsomes fractions isolated from human livers were incubated with GTX3 and GTX2 purified from contaminated mussels. After different incubation times, incubated samples were extracted and analyzed by HPLC with fluorescent on line detection and HPLC-MS analysis. The results revealed that GTX3 and GTX2, only when they were incubated with microsomal fraction and appropriated cofactors, showed to be enzymatic transformed in vitro. The glucuronidation of GTX3 and GTX2 followed typical Michaelis-Menten kinetics, resulting in apparent kinetic parameters of Km=39.4+/-0.24 microM and Vmax=6.0x10(-3) pmol/min/mg protein. In addition, the microsomes fraction also oxidized GTX3 and GTX2 into Gonyautoxin 4 (GTX 4) and Gonyautoxin 1 (GTX 1) resulting in 0.339x10(-3) pmol/min/mg protein. In conclusion, this study reports oxidation and glucuronidation of GTX3 and GTX2 when they are incubated with human liver microsomal fraction. The metabolism occurs via a glucuronidation reaction, the basis first step of biotransformation in human liver. Also it is showed that GTX4 and GTX1 came by biotransformation from GTX3 and GTX2 in humans. This data confirm human biotransformation found in human post-mortem fluid and tissue samples described previously. This data is the first evidence of in vitro glucuronidation of PST, given a metabolic pathway of detoxification and excretion of PST in human.

GTX 2/3 epimers permeate the intestine through a paracellular pathway.

The aim of this work was to typify the mechanisms involved in gonyautoxins intestinal permeability. For this purpose, permeability of gonyautoxins through intestinal epithelium, and their effect on transepithelial resistance was investigated in excised human jejunal segments. The isolated mucosa segments were mounted in a Ussing chamber and experiments performed under voltage-controlled conditions. Organic gonyautoxin cations were applied in the apical side and samples collected in the basolateral side. Results show that gonyautoxin 2/3 epimers (GTX 2/3) permeate the intestine through a paracellular pathway and, to reach the resolution of the technique we used, no evidence was found of any other transport mechanism involved in the process. A model was developed, according to which tight junctions undergo a toxin concentration and time-dependent change, while transepithelial resistance shows a modest decrease.