Oral Chronic Toxicity of the Safe Tetrodotoxin Dose Proposed by the European Food Safety Authority and Its Additive Effect with Saxitoxin.

Tetrodotoxin (TTX) is a potent natural toxin causative of human food intoxications that shares its mechanism of action with the paralytic shellfish toxin saxitoxin (STX). Both toxins act as potent blockers of voltage-gated sodium channels. Although human intoxications by TTX were initially described in Japan, nowadays increasing concern about the regulation of this toxin in Europe has emerged due to its detection in fish and mollusks captured in European waters. Currently, TTX is only regularly monitored in Dutch fishery products. However, the European Food Safety Authority (EFSA) has established a safety level of 44 µg/kg TTX as the amount of toxin that did not cause adverse effects in humans. This level was extrapolated considering initial data on its acute oral toxicity and EFSA remarked the need for chronic toxicity studies to further reduce the uncertainty of future toxin regulations. Thus, in this work, we evaluated the oral chronic toxicity of TTX using the safety levels initially recommended by EFSA in order to exclude potential human health risks associated with the worldwide expanding presence of TTX. Using internationally recommended guidelines for the assessment of oral chronic toxicity, the data provided here support the proposed safety level for TTX as low enough to prevent human adverse effects of TTX even after chronic daily exposure to the toxin. However, the combination of TTX with STX at doses above the maximal exposure level of 5.3 µg/kg body weight derived by EFSA increased the lethality of TTX, thus confirming that both TTX and paralytic shellfish toxins should be taken into account to assess human health risks.

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.

The Acute Toxicity of Tetrodotoxin and Tetrodotoxin⁻Saxitoxin Mixtures to Mice by Various Routes of Administration.

Tetrodotoxin (TTX) is a potent neurotoxin associated with human poisonings through the consumption of pufferfish. More recently, TTX has been identified in bivalve molluscs from diverse geographical environments, including Europe, and is therefore recognised as an emerging threat to food safety. A recent scientific opinion of the EFSA Panel on Contaminants in the Food Chain recognised the need for further data on the acute oral toxicity of TTX and suggested that, since saxitoxin (STX) and TTX had similar modes of action, it was possible that their toxicities were additive so could perhaps be combined to yield one health-based guideline value. The present study determined the toxicity of TTX by various routes of administration. The testing of three different mixtures of STX and TTX and comparing the experimentally determined values to those predicted on the basis of additive toxicity demonstrated that the toxicities of STX and TTX are additive. This illustrates that it is appropriate to treat TTX as a member of the paralytic shellfish group of toxins. Since the toxicity of TTX was found to be the same as STX by feeding, a molar toxicity equivalence factor of 1.0 for TTX can be applied.

Long-lasting, reversible and non-neurotoxic inactivation of hippocampus activity induced by neosaxitoxin.

BACKGROUND: Neosaxitoxin (NeoSTX) and related paralytics shellfish toxins has been successfully used as local anesthetic and muscle relaxants to treat a variety of ailments. The primary mechanism of action of these toxins occurs by blocking voltage-gated sodium channels with compounds such as TTX, lidocaine, or derivatives. However, most of these non-classical sodium channel blockers act with a reduced time effect as well as ensuing neurotoxicity. NEW METHOD: In this report, we show that the use of local NeoSTX injections inactivates the hippocampal neuronal activity reversibly with a by long-term dynamics, without neuronal damage. RESULTS: A single 10 ng/µl injection of NeoSTX in the dorsal CA1 region abolished for up to 48 h memory capacities and neuronal activity measured by the neuronal marker c-fos. After 72 h of toxin injection, the animals fully recover their memory capacities and hippocampal neuronal activity. The histological inspection of NeoSTX injected brain regions revealed no damage to the tissue or reactive gliosis, similar to vehicle injection. Acute electrophysiological recording in vivo shows, also, minimal spreading of the NeoSTX in the cerebral tissue. COMPARISON WITH EXISTING METHODS: Intracerebral NeoSTX injection showed longer effects than other voltage sodium channel blocker, with minimal spreading and no neuronal damage. CONCLUSION: NeoSTX is a new useful tool that reversibly inactivates different brains region for a long time, with minimal diffusion and without neuronal damage. Moreover, NeoSTX can be used as a valuable sodium channel blocker for many studies in vivo and with potential therapeutic uses.

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.

Acute Toxicities of the Saxitoxin Congeners Gonyautoxin 5, Gonyautoxin 6, Decarbamoyl Gonyautoxin 2&3, Decarbamoyl Neosaxitoxin, C-1&2 and C-3&4 to Mice by Various Routes of Administration.

Paralytic shellfish poisoning results from consumption of seafood naturally contaminated by saxitoxin and its congeners, the paralytic shellfish toxins (PSTs). The levels of such toxins are regulated internationally, and maximum permitted concentrations in seafood have been established in many countries. A mouse bioassay is an approved method for estimating the levels of PSTs in seafood, but this is now being superseded in many countries by instrumental methods of analysis. Such analyses provide data on the levels of many PSTs in seafood, but for risk assessment, knowledge of the relative toxicities of the congeners is required. These are expressed as "Toxicity Equivalence Factors" (TEFs). At present, TEFs are largely based on relative specific activities following intraperitoneal injection in a mouse bioassay rather than on acute toxicity determinations. A more relevant parameter for comparison would be median lethal doses via oral administration, since this is the route through which humans are exposed to PSTs. In the present study, the median lethal doses of gonyautoxin 5, gonyautoxin 6, decarbamoyl neosaxitoxin and of equilibrium mixtures of decarbamoyl gonyautoxins 2&3, C1&2 and C3&4 by oral administration to mice have been determined and compared with toxicities via intraperitoneal injection. The results indicate that the TEFs of several of these substances require revision in order to more accurately reflect the risk these toxins present to human health.

Low dose extended exposure to saxitoxin and its potential neurodevelopmental effects: A review.

Saxitoxin (STX) and its analogs, the paralytic shellfish toxins (PSTs), are a group of potent neurotoxins well known for their role in acute paralytic poisoning by preventing the generation of action potentials in neuronal cells. They are found in both marine and freshwater environments globally and although acute exposure from the former has previously received more attention, low dose extended exposure from both sources is possible and to date has not been investigated. Given the known role of cellular electrical activity in neurodevelopment this pattern of exposure may be a significant public health concern. Additionally, the presence of PSTs is likely to be an ongoing and possibly increasing problem in the future. This review examines the neurodevelopmental toxicity of STX, the risk of extended or repeated exposure to doses with neurodevelopmental effects, the potential implications of this exposure and briefly, the steps taken and difficulties faced in preventing exposure.

Neosaxitoxin in Rat Sciatic Block: Improved Therapeutic Index Using Combinations with Bupivacaine, with and without Epinephrine.

BACKGROUND: Neosaxitoxin (NeoSTX) is a site-1 sodium channel blocker undergoing clinical trials as a prolonged-duration local anesthetic. Rat sciatic block and intravenous infusion models were used to assess efficacy and local and systemic toxicities for NeoSTX in saline (NeoSTX-Saline), bupivacaine (Bup), and their combination (NeoSTX-Bup). Exploratory studies evaluated the effects of addition of epinephrine to NeoSTX-Bup (NeoSTX-Bup-Epi). METHODS: Rats received percutaneous sciatic blocks with escalating doses of NeoSTX-Saline or NeoSTX-Bup. Sensory-nocifensive block was assessed using modified hotplate and Von Frey filaments. Motor-proprioceptive function was assessed by extensor postural thrust. Nerves were examined histologically after 7 days and scored on the Estebe-Myers scale. Median lethal dose was estimated for NeoSTX-Saline and in combinations. Accidental intravenous overdose was simulated in isoflurane-anesthetized, spontaneously breathing rats receiving NeoSTX-Saline (n = 6), Bup (n = 7), or NeoSTX-Bup (n = 13), with respiratory, hemodynamic, and electrocardiographic endpoints. Additional groups received blocks with NeoSTX-Bup-Epi (n = 80). Investigators were blinded for behavioral and histologic studies. RESULTS: NeoSTX-Bup produced more prolonged sensory and motor block compared with NeoSTX-Saline or Bup. NeoSTX-Bup-Epi further prolonged median time to near-complete recovery for 3 µg/kg NeoSTX-Bup (hotplate: 48 vs. 6 h, P < 0.001). With sciatic injections, addition of Bup did not worsen the systemic toxicity (median lethal dose) compared with NeoSTX-Saline. Intravenous NeoSTX-Saline infusion had significantly longer times to apnea, first arrhythmia, and asystole compared with Bup (P < 0.001 for each). Histologic injury scores overall were low for all groups, with median scores of 0 (interquartile range, 0 to 0) on a 5-point scale. CONCLUSION: NeoSTX-Bup and NeoSTX-Bup-Epi hold promise for prolonged-duration local anesthesia.

A Phase 1, Dose-escalation, Double-blind, Block-randomized, Controlled Trial of Safety and Efficacy of Neosaxitoxin Alone and in Combination with 0.2% Bupivacaine, with and without Epinephrine, for Cutaneous Anesthesia.

BACKGROUND: Neosaxitoxin (NeoSTX) is a site-1 sodium channel blocker that produces prolonged local anesthesia in animals and humans. Under a Food and Drug Administration-approved phase 1 Investigational New Drug trial, the authors evaluated safety and efficacy of NeoSTX alone and combined with 0.2% bupivacaine (Bup) with and without epinephrine. METHODS: The authors conducted a double-blind, randomized, controlled trial involving healthy male volunteers aged 18 to 35 yr receiving two 10-ml subcutaneous injections. Control sites received Bup. In part 1, active sites received (1) 5 to 40 µg NeoSTX+Saline (NeoSTX-Saline), (2) 5 to 40 µg NeoSTX+Bup (NeoSTX-Bup), or (3) placebo (Saline). In part 2, active sites received 10 or 30 µg NeoSTX+Bup+Epinephrine (NeoSTX-Bup-Epi) or placebo. Primary outcome measures were safety and adverse events associated with NeoSTX. Secondary outcomes included clinical biochemistry, NeoSTX pharmacokinetics, and cutaneous hypoesthesia. RESULTS: A total of 84 subjects were randomized and completed the two-part trial with no serious adverse events or clinically significant physiologic impairments. Perioral numbness and tingling increased with NeoSTX dose for NeoSTX-Saline and NeoSTX-Bup. All symptoms resolved without intervention. NeoSTX-Bup-Epi dramatically reduced symptoms compared with other NeoSTX combinations (tingling: 0 vs. 70%, P = 0.004; numbness: 0 vs. 60%, P = 0.013) at the same dose. Mean peak plasma NeoSTX concentration for NeoSTX-Bup-Epi was reduced at least two-fold compared with NeoSTX-Saline and NeoSTX-Bup (67 ± 14, 134 ± 63, and 164 ± 81 pg/ml, respectively; P = 0.016). NeoSTX-Bup showed prolonged cutaneous block duration compared with Bup, NeoSTX-Saline, or placebo, at all doses. Median time to near-complete recovery for 10 µg NeoSTX-Bup-Epi was almost five-fold longer compared with Bup (50 vs. 10 h, P = 0.007). CONCLUSION: NeoSTX combinations have a tolerable side effect profile and appear promising for prolonged local anesthesia.

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.

Chronic toxicity study of neosaxitoxin in rats.

Neosaxitoxin (NeoSTX) is a specific reversible blocker of voltage gated sodium channels on excitable cells. In the last decade, it has been tested in a number of interesting clinical trials, however there is still little information available on mammalian toxicity. Rats were treated for 12 weeks with doses of 1, 3 or 6 µg/kg of subcutaneous NeoSTX. At weeks 12 and 17, animals were sacrificed and blood samples collected for hematological and biochemical analysis. Organs were harvested for weight determination and histopathological assessments. The lowest acute toxicity via the intraperitoneal (ip) route was (30.35 µg/kg) and there was no significant difference between intramuscular and subcutaneous routes (11.4 and 12.41 µg/kg). The NeoSTX adiministration did not produce lethality at week 12 and after five weeks of suspension. NeoSTX 6 µg/kg ip produced reductions (p < 0.05) in body weight and food intake, and increased blood level of total and direct bilirubin, GGT and SGOT at week 12; all of these were reversed in the recovery period. NeoSTX 1 and 3 µg/kg ip did not show significant changes with the control group. Histopathological presentations were normal in all groups. This study revealed that NeoSTX is safe in vivo, giving a reliable security margin for its use like a local anesthetic.

Apoptosis of hemocytes from lions-paw scallop Nodipecten subnodosus induced with paralyzing shellfish poison from Gymnodinium catenatum.

The toxic dinoflagellate Gymnodinium catenatum produces paralyzing shellfish poisons (PSPs) that are consumed and accumulated by bivalves. Previously, we recorded a decrease in hemocytes 24h after injection of PSPs (gonyautoxin 2/3 epimers, GTX2/3) in the adductor muscle in the lions-paw scallop Nodipecten subnodosus. In this work, qualitative and quantitative analyses, in in vivo and in vitro experiments, revealed that the lower count of hemocytes results from cells undergoing typical apoptosis when exposed to GTX 2/3 epimers. This includes visible morphological alterations of the cytoplasmic membrane, damage to the nuclear membrane, condensation of chromatin, DNA fragmentation, and release of DNA fragments into the cytoplasm. Induction of apoptosis was accompanied by phosphatidylserine exposure to the outer cell membrane and activation of cysteine-aspartic proteases, caspase 3 and caspase 8. Addition of an inhibitor of caspase to the medium suppressed activation in hemocytes exposed to the toxins, suggesting that cell death was induced by a caspase-dependent apoptotic pathway. The results are important for future investigation of the scallop's immune system and should provide new insights into apoptotic processes in immune cells of scallops exposed to PSPs.

Acute toxicities of saxitoxin, neosaxitoxin, decarbamoyl saxitoxin and gonyautoxins 1&4 and 2&3 to mice by various routes of administration.

Saxitoxin and its derivatives, the paralytic shellfish toxins (PSTs), are known to be toxic to humans, and maximum permitted levels in seafood have been established by regulatory authorities in many countries. Until recently, the mouse bioassay was the reference method for determining the levels of these toxins in seafood, but this has now been superseded by chemical methods of analysis. The latter methods are able to determine the levels of many PSTs in shellfish, but for risk assessment an estimate of the relative toxicities of the individual components of the PST mixture is required. The relative toxicities are expressed as "Toxicity Equivalence Factors" (TEFs). At present, TEFs are based on relative specific activities in the mouse bioassay, rather than on acute toxicity determinations, as measured by median lethal doses (LD50s). In the present study, the median lethal doses of saxitoxin, neosaxitoxin, decarbamoyl saxitoxin and equilibrium mixtures of gonyautoxins 1&4 and gonyautoxins 2&3 have been determined by intraperitoneal injection, gavage and feeding. The results indicate that specific activities in the MBA do not consistently correlate with acute toxicities by any of the routes of administration, and TEFs, particularly for neosaxitoxin, require revision.

Prolonged nerve blockade delays the onset of neuropathic pain.

Aberrant neuronal activity in injured peripheral nerves is believed to be an important factor in the development of neuropathic pain. Pharmacological blockade of that activity has been shown to mitigate the onset of associated molecular events in the nervous system. However, results in preventing onset of pain behaviors by providing prolonged nerve blockade have been mixed. Furthermore, the experimental techniques used to date to provide that blockade were limited in clinical potential in that they would require surgical implantation. To address these issues, we have used liposomes (SDLs) containing saxitoxin (STX), a site 1 sodium channel blocker, and the glucocorticoid agonist dexamethasone to provide nerve blocks lasting ~1 wk from a single injection. This formulation is easily injected percutaneously. Animals undergoing spared nerve injury (SNI) developed mechanical allodynia in 1 wk; nerve blockade with a single dose of SDLs (duration of block 6.9 ± 1.2 d) delayed the onset of allodynia by 2 d. Treatment with three sequential SDL injections resulting in a nerve block duration of 18.1 ± 3.4 d delayed the onset of allodynia by 1 mo. This very prolonged blockade decreased activation of astrocytes in the lumbar dorsal horn of the spinal cord due to SNI. Changes in expression of injury-related genes due to SNI in the dorsal root ganglia were not affected by SDLs. These findings suggest that formulations of this kind, which could be easy to apply clinically, can mitigate the development of neuropathic pain.

Respiratory, neuromuscular, and cardiovascular effects of neosaxitoxin in isoflurane-anesthetized sheep.

BACKGROUND: Neosaxitoxin (NeoSTX) is a potent site-1 sodium-channel blocker being developed as a local anesthetic. Doses of 100 µg have been used by local infiltration in anesthetized adult humans without adverse effect. We hypothesized that similar doses could cause significant respiratory, neuromuscular, and cardiovascular impairment and sought to test this hypothesis in sheep. METHODS: Procedures were approved by the Institutional Animal Care and Use Committee. In neuromuscular/respiratory experiments, 33 intubated, isoflurane-anesthetized sheep were randomized to 6 NeoSTX treatment groups: saline control, 1 µg/kg subcutaneous (SC), 1 µg/kg intravenous (IV), 2 µg/kg SC, 2 µg/kg SC with bupivacaine 0.25%, and 3 µg/kg SC. Primary outcome measures were doxapram-stimulated inspired volume (DSIV) and quantitative limb acceleration. In cardiovascular experiments, 8 sheep received escalating IV doses of NeoSTX (1, 2, and 3 µg), with hemodynamic and electrocardiographic measurements. Data were analyzed using repeated-measures analysis of variance with post hoc Bonferroni-corrected comparisons. RESULTS: NeoSTX 1 µg/kg IV and SC produced no significant reduction in DSIV or limb acceleration compared with baseline. NeoSTX 2 µg/kg SC produced clinically mild reduction in twitch and DSIV; animals recovered well postoperatively. Coadministration of bupivacaine did not worsen these effects. NeoSTX 3 µg/kg produced severe and prolonged impairment of DSIV and limb acceleration. Escalating IV doses of NeoSTX produced mild decrements in heart rate, systemic arterial pressure, and systemic vascular resistance; cardiac output was maintained. Transient interventricular conduction delay occurred without cardiac arrest or ventricular ectopy. CONCLUSIONS: In our sheep model, neuromuscular, respiratory, and cardiovascular effects of NeoSTX were dose dependent and mild using the dose range anticipated for clinical use.

Alterations of 3,4-dihydroxyphenylethylamine and its metabolite 3,4-dihydroxyphenylacetic produced in rat brain tissues after systemic administration of saxitoxin.

We have measured the dopamine levels in some discrete rat brain regions after acute intraperitoneal administration of saxitoxin (STX). STX is one of the several toxins that causes paralytic shellfish poisoning (PSP). PSP is a serious public health concern through the world. Certain dinoflagellates are able of producing STX, a powerful neurotoxic compound, which blocks the voltage sensitive sodium channels, entailing to the appearance of the main symptoms of poisoning by PSP: muscular paralysis and respiratory depression. The goal in this study was to analyze the effect of STX on dopamine levels in discrete rat brain regions after its acute intraperitoneal administration. Different experimental periods were analyzed for STX doses (5 and 10 µg kg(-1) body weight). With low dose, experimental periods were: 30, 60 and 120 min. With high dose, experimental period was just 30 min. At the end of each experimental period, animals were sacrificed by cervical dislocation. Brains were removed and dissected in: hypothalamus, striatum, midbrain, brain stem, right and left hemispheres. This is to our knowledge, the first report in which a sublethal dose of STX administered intraperitoneally results in an acute alteration of dopamine (DA) production and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC).

Comparison of neosaxitoxin versus bupivacaine via port infiltration for postoperative analgesia following laparoscopic cholecystectomy: a randomized, double-blind trial.

BACKGROUND AND OBJECTIVES: Wound infiltration with available local anesthetics generally provides analgesia for less than 8 hrs. The site 1 sodium-channel toxin neosaxitoxin (neoSTX) produced analgesia for over 24 hrs in animals and human volunteers. In this randomized, double-blind trial, we examined the postoperative course of patients undergoing laparoscopic cholecystectomy under a standardized general anesthesia with wound infiltration using either neoSTX or bupivacaine. We hypothesized that neoSTX would reduce pain compared with bupivacaine at 12 hrs postoperatively. METHODS: Patients received preincisional infiltration of laparoscope entry sites with 20 mL containing either neoSTX (total dose, 100 µg) or bupivacaine 0.25% (total dose, 50 mg). The primary outcome measure was the visual analog pain score at 12 hrs postoperatively. Secondary outcomes included repeated pain scores at rest and with movement,analgesic use, functional recovery, and adverse effects. Groups were compared using Mann-Whitney U tests for pain scores, Fisher exact test for proportions of patients with severe pain and complete analgesia, and Kaplan-Meier curves for time to full recovery. RESULTS: Among 137 subjects, 69 were randomized to neoSTX and 68 to bupivacaine. Median pain scores at rest and with movement 12 hrs postoperatively were lower in the neoSTX group compared with the bupivacaine group (P<0.01). Additional pain measures and recovery parameters also favored neoSTX. No serious adverse events occurred,and no adverse events were more frequent in the neoSTX group. CONCLUSIONS: NeoSTX shows promise as a long-acting local anesthetic. Future studies will examine dose response, combination formulations, and safety with dose escalation.