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Review Article ISSN-2572-7656

Phycotoxins of the Paralytic Shellfish Poison: Clinical Applications Néstor Lagos Biochemical Membrane Laboratory, Department of Physiology and Biophysics, Faculty of Medicine, University of Chile.

exhibit potent biological activities. They are secondary metabolites, Introduction which are not vital to the plain metabolism and growth of the A drug is defined as an agent intended for use in the diagnosis, organism, but they are present in constrained taxonomic groups. mitigation, treatment, cure, or prevention of disease in humans or in Among the many algal secondary metabolites that have been identi- other animals. Certainly, the vast array of effective medical agents fied, an important number are potent biotoxins responsible for a available today represents one of our greatest scientific accompli- wide array of human illnesses. shments. It is difficulty to conceive our civilization devoid of these Four major illnesses associated with microalgae biotoxins and remarkable and beneficial agents. Drug, in the form of vegetation and with shellfish poisoning have been described: Paralytic shellfish minerals, have existed longer than humans. Human disease and the poisoning, Diarrheic shellfish poisoning, Amnesic shellfish poisoning instinct to survive have, through the ages, led to their discovery. and Neurotoxic shellfish poisoning [6, 7]. Paralytic shellfish poiso- Throughout history, the knowledge of drug and their application to ning, which shown as primary clinical symptom acute paralytic disease has always meant power. illness, poses the most serious threat to public health due to its high Searching for potential clinical applications, poisonous substances mortality rate in mammals [4]. from plants, animals and microorganisms have been tested in studies Paralytic shellfish poisoning(PSP), is the most widespread algae on animal physiology. The most well-known example is the clinical derived shellfish poisoning worldwide [4]. The PSP toxins are a use of botulin toxin type A, which has been shown to be useful in group of over 28 structurally related imidazoline guanidinium several therapeutic approaches [1]. derivatives, non-protein phycotoxins with low molecular weight The first definition of a “Red Tides” was done by Okamura [2] in ranging from 280 to 450 Daltons. They have a common chemical Japan since this phenomenon is frequently occurrence there, he skeleton (3, 4, 6-trialquil tetrahidropurine) that makes them defined as follows: “Akashio ( in Japanese) refers to the water hydrophilic, so totally soluble in water [8]. According to the net colour change due to outbreaks of microscopic plankton which can charge that these toxins show at pH 7.0, they can classified in three sometimes cause the death of fish and others animals, irrespective of major groups: (1) (STXs) with a net charge of +2; (2) the colour [2]. The expression “red tide” become common in Japan gonyaulatoxins (GTXs) group with net charge of +1, and (3) N- during the latest half of the 1960s. The description “red tide” is visual sulfocarbamoyl-11-hydroxysulfate toxins (Cs) with net charge 0. The and not scientific, and the phenomenon describes by Okamura is was the first PSP toxin described and its structure worldwide known as “water blooms” and more accurate Harmful established by x-ray analysis [9, 10] it is the most well know and Algae Blooms (HABs), since they produce serious negative impacts in studied. Also, it is the most frequently found commercially available. marine environments. These occurrences of toxic or harmful Nevertheless, (GTXs) and specially GTX2/3 and microalgae represent a significant and seemingly expanding threat to GTX1/4 epimers are the most abundant in molluscs extract samples human health, fishery resources, and marine ecosystems throughout and they account for the shellfish high toxicity in Chile and the world [3]. worldwide [4, 5, 11, 12]. A number of marine microalgae species produces phycotoxins The organisms that are recognized as primary sources of PSP that are responsible for massive fish kill and seafood-related poiso- toxins include three morphologically distinct genera of dinoflage- ning in humans [4, 5]. The symptoms are often neurological and/or llates, Alexandrium sp., Pyrodinium sp., and Gymnodinium sp [11] gastrointestinal and linked to an altered cellular excitability. Microalgae and six species of blue-green alga, Aphanizomenon flos-aquae Ralfs primary producers that make up the base of both marine and fresh ex Bornet & Flahault [13], Lyngbyawollei Farlow ex Gormont [14] water food webs. Some of them also produce unusual compounds that from North America; Anabaena circinalis Rabenhorst ex Bornet and Flahault [15] from Australia; Cylindrospermopsisraciborskii (Wolo- synska) Seenayya and Subba Raju [16] and Microcystis aeruginosa Kutzing [17] from Brazil; Planktothrix Anagnostidis and Komáreck sp. [18] from Italy, and Aphanizomenonflos-aquaeRalfs ex Bornet & *Address for Correspondence: Néstor Lagos, Biochemical Membrane Flahault [19] from Portugal. The last two were found in European Laboratory, Department of Physiology and Biophysics, Faculty of Medicine, continental freshwaters and the two species reported from Brazil are University of Chile, Independence 1027, Santiago, Chile, E-mail: the only two described in South American freshwater. [email protected] Received: January 28, 2018; Accepted: March 03, 2017; Published: March 05, In Chilean littoral, these PSP toxins are produced by the 2018 dinoflagellate Alexandrium catenella, which is filtrated by bivalve

Clin Pharmacol Transl Med, 2018 Volume 2 (2): 89 - 100 Lagos N. Phycotoxins of the Paralytic Shellfish Poison: Clinical Applications. Clin Pharmacol Transl Med. 2018; 2(2): 89-100 molluscs, concentrating the toxins in high amount. The PSP toxins can be purified starting from high-contaminated shellfish collected in the austral southern Chilean fjords. Actually, in these Patagonian fjords the shellfish highest toxicity ever reported [20] has been described. Until now, all the clinical applications using PSP toxins have been performed merely in the University of Chile Clinical Hospital in Santiago, Chile. Here, due to a pioneer basic science - clinical collaboration the therapeutic usage of PSP toxins had been established. The therapeutics properties of these biotoxins after local infiltration showed to be safe and effective. This report describes the therapies, involving local infiltration of PSP toxins, characterized by immediate pain relief and muscle relaxation, both effects displayed in minutes after biotoxins infiltration with amazing successful clinical results. The data published in those clinical trials will be analysed and discussed in this chapter. The molecular mechanism underlying the PSP Toxins clinical effects The high toxicity of the PSP toxins is due to the reversible binding to a site receptor on the voltage-gated sodium channel on excitable cells [21, 22], blocking neuronal transmission and causing mammals death by respiratory arrest and cardiovascular shock [23-28]. PSP toxins bind with high affinity (Saxitoxin Kdlower than 2 nM) to site 1 on the voltage-dependent sodium channel, inhibiting channel opening. The voltage- dependent sodium channels play a key role in neurotransmission at both neuronal synapses and neuromuscular junctions. Since all the PSP toxins share two guanidine groups, in carbon 2 and 8; they show positive net charge with a protonic dissociation at pKa 8.22 and 11.28 [28]. The ammonium quaternary groups in their chemical structure provide them high polarity, so they cannot go across the blood-brain barrier [28]. Consequently, their main physiological effect is linked to the blocking action at axonal level impeding both, nerve impulse propagation and neuronal transmition over the neuromuscular junction. Therefore, when they are applying locally, two clinical activities are manifested simultaneous: (i) the control of pain (anesthesic activity) and (ii) the control of muscle hyperactivity (relaxant effect). In this review, we will discuss the local application of PSP toxins, which produces muscular flaccid paralysis and anesthesia for periods that are dose dependent.

Clinical trials published from 2004 to 2017 Authors Year Title Details Lagos, N, García, 2004 Paralytic Shellfish Poison: toxins that can Proceeding Chemical and C., Lattes, K, Lagos, kill and heal Biological Medical M., et al.[29] Treatment Symposium, Switzerland

Garrido, R., Lagos, 2004 The 2/3 epimers reduces anal Biol. Research, 37: 395- N., Lattes, K, et tone when injected in the anal sphincter of 403. al.[30] healthy adults Garrido, R., Lagos, 2005 Gonyautoxin: New treatment for healing Diseases of the Colon & N., Lattes, K, et al. acute and Chronic anal fissures. Rectum 48: 335 – 343. [31] Garrido, R., Lagos, 2005 New toxin treatment for anal fissures. Research Highlights, N., Lattes, K, et Nature Clinical Practice al.[32] Gastroenterology & Hepatology. 3, 2: 126-127. Garcia, C., Lagos, 2005 Human intoxication with Paralytic Biological Research 38: M., Truan, D, et Shellfish Toxins: Clinical parameters and 197-205. al.[33] toxin analysis in plasma and urine. Rodriguez-Navaro 2006 Intrasphincteric neosaxitoxin injection: Am J Gastroenterol. Nov; AJ, Lagos N, Lagos evidence of lower esophageal sphincter 101 (11): 2667-2668. M, et al.[34] relaxation in achalasia.

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Rodriguez-Navarro, 2007 Neosaxitoxin as a Local Anesthesic: Anesthesiology 106: 339- A.J., Lagos, N., Preliminary Observations from a First 345. Lagos, et al.[35] Human Trial. Garrido, R., Lagos, 2007 Treatment of Chronic Anal Fissure by Colorectal Disease 9: 619- N., Lagos, M. [36] Gonyautoxin. 624. Torres R. Pizarro, 2007 GTX 2/3 epimers permeate the intestine Journal of Toxicological L., Csendes, A, et through a paracellular pathway. Sciences, 32: 241-248. al.[37] García, C., 2009 Evidence of in vitro glucuronidation and Toxicon, 53: 206-213. Rodriguez-Navarro, enzymatic transformation of paralytic A., Días, J.C., et shellfish toxins by healthy human liver al.[38] microsomes fraction. Lattes, K., 2009 Local Infiltration of Gonyautoxin is safe Neurological Research, 31: Venegas,P., Lagos, and effective in treatment of Chronic 208-223. N, et al.[39] Tension-Type Headache. Rodriguez-Navarro, 2009 Potentiation of Local Anesthetic Activity Neurotoxicity Research, A., Lagos, M., of Neosaxitoxin with or 16: 408 - 415. Figueroa, C., et al Epinephrine: Development of a Long- [40]. Acting Pain Blocker. García, C., Barriga, 2010 Route of metabolization and detoxication Toxicon, 55: 135-144. A., Díaz, J.C., et of Paralytic Shellfish Toxins in humans. al.[41] V. Manríquez., 2015 First evidence of Neosaxitoxin as a long- IntUrogynecol J. DOI D.Castro., R. acting pain-blocker in Bladder Pain 10.1007/s00192-014-2608- Guzmán, et al.[42] Syndrome. 2. (*) Hinzpeter J., 2016 Gonyautoxins: First evidence in pain Toxicon, 119: 180-185. Barrientos M., management in total knee arthroplasty. Zamorano A, et al.[43]

(*): This manuscript was recommended by F1000PRIME 2015 as being of special significance in the field.

Treatment of chronic anal fissures Anal fissure is a cut or crack in the anal canal that may extend from the mucocutaneous junction to the dentate line, which most commonly occurs in the posterior midline.Once the tear occurs, it begins a cycle leading to repeated injury. The exposed internal sphincter muscle beneath the tear goes into spasm, which pulls the edges of the fissures apart impeding the healing of the wound. This cycle leads to the development of a chronic anal fissure.This is a common problem that causes substantial morbidity with roughly equal incidence in both genders and shows great reluctance to heal without intervention [44, 45]. The major symptoms of chronic anal fissure are permanent pain, intense pain during defecation, lastingat least four hours, bright blood on the toilet paper and on the surface of stools, as well as sphincter cramps [44]. The elevation of anal pressure in patient with chronic anal fissure may result from increased tone of the internal anal sphincter, which is clearly observed manometrically, the resting anal pressure being elevated in fissure patients [46]. Therefore, most of the therapies are focused to diminish the anal tone.

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Citation: Swerdlow RH, Lyons KE, Khosla SK, Nashatizadeh M, Pahwa R. A Pilot Study of Oxaloacetate 100 mg Capsules in Parkinson ’s disease Patients. J Parkinsons Dis Alzheimer Dis. 2016;3(2): 4.

*Address for Correspondence:Leandro Bueno Bergantin, Rua Pedro de Toledo, 669 – Vila Clementino, São Paulo – SP, Brazil, CEP: 04039-032. Fax: 1-913-588-0681; E-mail: [email protected] Lagos N. Phycotoxins of the Paralytic Shellfish Poison: Clinical Applications. Clin Pharmacol Transl Med. 2018; 2(2): 89-100

For this pathology, we proposed the use of PSP toxins to produce the internal sphincter muscle relaxation, in order to end with the Injury Inflammation ischemia allowing the capillary to start epithelized and at the same (Anal Fissure) time to block pain, since PSP toxins are potent local anesthesic. Since, both effect occurs simultaneous, we expect subsequently to impro- vethe quality of patient life from the same instant that we infiltrate with PSP toxins [30-32, 47]. As a result, the epimers Gonyautoxin 2/3 (GTX 2/3), the most abundant PSP toxin found in southern Chilean shellfish (Mytiluschi- lensis) was chosen for a first clinical trial. The phycotoxins were purified from highly PSP toxin contaminated shellfishes collected in Chilean Patagonia fjords [4]. Sphincter Spasm Pain The anal fissure studies were performed at the Coloproctology Section, Surgery Department, Clinical Hospital-University of Chile, Santiago, Chile. All the trials complied with the Declaration of Figure 1: The vicious loop of chronic anal fissure (injury, inflamma- Helsinki regarding medical research involving human volunteers, tion, pain, and sphincter spasm). approved by an-institutional reviewing board at the Clinical Hospital Ethics Committee and the Public Health Institute, Santiago, Chile. was with 50 patients [31] and the second with 23 patients [36]. The Public Health Institute is the equivalent to the USA, Food and The only difference between them was on the frequency of infiltra- Drugs Administration (FDA) in Chile. The design and purpose of tion. In the first trial, the infiltration was repeated after 7 days and in each study, as well as the potential risks for the participants were the second trial, infiltration was repeated after 4 days. discussed before enrollment. Their written informed consent was always obtained. In both trials, adults, between 18 and 70 years old, diagnosed with symptomatic anal fissures were enrolled. The recruited patients In general, each toxin doses consisted of a sterile solution of 100 received clinical examination, including digital evaluation, units of GTX 2/3 epimers in 1.0 ml total volume of 0.9% NaCl, proctoscopy and questionnaires to evaluate the symptoms. Anorectal without preservatives. An insulin syringe with a 25-gauge needle was manometries were performed before and 4 minutes after the toxin used for the injection. One unit of the paralyzing toxin activity infiltration. The dose was infiltrated in a total volume of 1 millilitre corresponds to the amount of toxin enough to block neuromuscular into both sides of the anal fissure, within the internal anal sphincter contraction of a mouse leg crural bicep for 1.5 to 2.0 hours. (0.5 ml in each side). The patients were clinically evaluated 4, 7, 14, In the first clinical trial, the effect of GTX 2/3 was studied over 21 and 28 days after the beginning of the treatment. The infiltration the anal tone of healthy adults, showing a relaxing effect in all pain scores and that two minutes after the injection were evaluated by participants. Moreover, manometric recording showed a significant a VAS scale from 1 to 10, being 10 the maximum pain value. Long- decrease in anal maximal voluntary contraction pressure after the term outcomes were determined at a14 months follow-up. In both toxin infiltration. The post-injection electromyography showed that studies the inclusion criteria were (i) evidence of anterior, posterior the muscle activity was abolished practically immediately after the or both circumscribed ulcers; (ii) induration of the edges, and (iii) injection [30]. No participant suffered any adverse events or negative exposure of horizontal fibres of internal anal sphincter, with side effects during or after the infiltration. The clinical laboratory tests symptoms of post defecation or permanent pain, bleeding, or both. performed before and after the infiltration did not show any Patients with fissure history of more than two months were significant changes. An important finding was that neither flatus nor considered as chronic anal fissure patients. The exclusion criteria faecal incontinence were observed. Furthermore, the ano-rectal were: (i) patients younger than 18 or older than 70 years; (ii) pregnancy, inhibitory and ano-cortical reflexes remained functional in all parti- and (iii) fissure associated with other pathological condition, such as cipants, indicating that the infiltration blocked muscle hyperactivity, haemorrhoids, fistula or anal abscesses. Both clinical trials conside- but leaving enough strength for physiological functioning [30]. red pain relief and total fissure epithelisation as primary endpoints. That study concluded that local intramuscular injection of GTX In the first trial, total remission of acute and chronic anal fissures 2/3 epimers into the anal internal sphincter produced immediate was achieved in most of the cases, 98% of the patients healed at a sphincter relaxation, decreasing the pressure produced by voluntary mean time of 17.6 ± 9 days. In the second trial, total remission was contraction. Hence, the effectiveness and safety of PSP toxin injection achieved earlier, with a mean time of healing of 8.2 ± 2.4 days, in humans is shown for the first time. Thereafter, we were ready to providing a more efficient treatment, since the patients healed test the drug for anal fissure in patients. practically in one week. The protocol used for the second trial was The theoretical background of the study refers to a temporary then proposed to patients. Indeed, the patients included in the first pharmacological immobilization of the anal sphincter for eliminating trial complained that the drug effect did not cover the full period of sphincter spasm, leading to the healing of the wound. This is the seventh days between the injections. critical step for treating and healing anal fissure, interrupting the The first clinical trial was initially proposed as a randomized, vicious loop of chronic anal fissure: the crack in the anal canal double-blind trial, for which the patients were consecutively recruited (injury), inflammation, pain and internal sphincter spasm. upon arrival to the Coloproctology Section, diagnosed and infiltrated In order to test the effect of these biotoxins, blocking pain and with toxin or placebo solution (toxin-free 0.9 % NaCl solution). The relax the internal sphincter, two clinical trials were performed. The first double-blind trial was opened for humanitarian and ethical reasons,

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since after three weeks of treatment the patients injected with toxin infiltration in chronic anal fissure were both effective and safe, but also were healthy and the ones injected with placebo showed no they showed faster recovery and unexpansive massive ambulatory improvement or worsening of their clinical condition. Moreover, 100 treatment, that could be the solution for those countries where percentages of the patients injected with the phycotoxin showed there is not enough surgeons available. immediate post injection anal sphincter relaxation, which was detected by digital examination and anorectal manometry. This effect Treatment of chronic tension-type headache was not observed following placebo injection, making unnecessary to According to the International Headache Society, tension-type keep the patients suffering for three or four weeks, to just keep the headache (TTH) is essentially defined as bilateral headache of a double-blind protocol. The double-blind protocol was opened with pressing or tightening quality that lacks a known medical cause. TTH the authorization of the Chilean Public Health Institute and the is classified as episodic if it occurs on less than 15 days a month, and as Hospital Ethic Committee. chronic if it occurs with larger frequency, meeting the International All patients declared to feel anaesthesia after the infiltration, and Headache Society diagnostic criteria (International Headache Society relieve from the intense anal pain. Thus, the life quality of the patients ICH-10 guide for headaches, 1997). was immediately and significantly improved. During the digital This clinical trial was designed to evaluate the therapeutic examination, coloproctologists detected an immediate anal tone properties of Gonyautoxin 2/3 epimers local infiltration in patients reduction, which persisted for at least five days. None flatus or faecal with chronic TTH, to propose a safe and effective alternative to incontinences were observed, explained by the full preservation of current treatment [39]. A study was initially proposed as a randomi- anal-rectal inhibitory and anal-cortical reflexes. Bleeding stopped in zed, double-blind trial in which the patients were consecutively all patients within 48 hours after infiltration and at the first clinical recruited upon arrival to the Hospital Neurology Clinic, where the control (4 days after), the patients showed epithelisation of the lesion, patients were diagnosed and randomly injected with either toxin or a with only modest pain when defecating. At the second clinical placebo solution. A randomized table generated by computer was used examination the lesion was totally epithelized with a healthy scar. to designate placebo or toxin infiltration for each patient. Due to the As a result, Gonyautoxin local infiltration into the internal anal immediate clinical effect produced by the gonyautoxin treatment, sphincter produced a temporary pharmacologic immobilization of immediately instantly detected by electromyography (EMG) after the the anal sphincter muscle that eliminated sphincter spasm, the critical infiltration, the study was modified and continued as an open label step, breaking the vicious circle of damage, inflammatory, pain and study. Two major effects could not be maintained in the blinding as sphincter spasm, consequently healing the anal fissure. PSP toxins initial proposed by the study: (i) a muscle relaxant effect detected infiltrations showed an outstanding efficacy and safety in anal fissure immediately by the physicians injecting the toxin, and (ii) a dropping treatment representing a new therapeutic approach. Due to its in the pain scores, referred by the patients 5 minutes post injection, efficacy and low cost it should be preferred to any other which was not observed in the placebo group. Both clinical effects pharmacologic treatment or surgery. Surgery implies permanent were only observed after the toxin infiltration and never following flatus and/or faecal incontinence risk, and an average of seven weeks placebo. Thus, 27 patients with chronic TTH were locally infiltrated for healing time, irreversible sphincter damage and costly with a gonyautoxin dose of 50 micrograms in 2 ml. The infiltration hospitalization. The present 4 days protocol showed to be very was applied intramuscularly in 10 sites considered as pain trigger efficient, shortening the healing time and the patient perception of points according to a fixed infiltration protocol (200 micro liters per healing. Actually, the authors recommend a safe three injections injection). EMG recording was performed before and immediately treatment: one as a starting infiltration (cero time), then twice every 4 after infiltration. days, in total 8 days of treatment. The TTH diagnosis was performed by neurologists according to About the potential intoxication that can occur with the dose the International Headache Society criteria and the inclusion criteria implemented (20 micrograms per patients), this is far below the safe were: (i) patients with headache of a pressing or tightening quality limits of international official regulations who allow consumption of with episodic frequency above 15 days. (ii) patients refractory to any shellfish with a safe limit of 80 micrograms of STX equivalent to 100 other conventional treatment, such as orally administered analgesics, grams of shellfish meat (Fish and Fishery Products Hazards and systemic muscular relaxant, corticoids and antidepressants like Controls Guidance 2006). , the gold standard treatment [48, 49]. All patients had been previously treated and controlled at the University Hospital This pathology has a prevalence of 1.2 to 1.5% of the total Neurology Clinic over 2 years, with symptom lasting over 3 years. population between 16 to 80 years old of both sex in developed countries (USA, Japan and Europe). In case of Chile, we have a The exclusion criteria were: (i) pregnancy; (ii) use of prophylactic similar data, since our population is around 17 million of people we headache treatment a month prior the infiltration; (iii) myasthenic should have around 250,000 patients wander with this disease. In any syndromes; (iii) muscular dystrophies; (iv) inflammatory myopathies; underdeveloped country it should be similar, for that reason, this (v) acute and chronic polyneuropathies, and (vi) use of anticoagulant pathology is also a major social problem because the gold standard in treatment, terminal illnesses (AIDS, cancer), drugs or abuse, or this pathology is a sphicterotomy surgery and normally in those psychotropic substances 24-hour before infiltration. The end-points countries the surgeons that practice this surgery are scarce. All the were: (a) reporting a control of the acute episode of pain at the patients that participate in our clinical trials, associated to anal moment of infiltration, defined as a 30% drop from the original pain fissure, belonged to the last economic quintile and they did not have score, and (b) prophylactic effect, escalation in the day number health security. They have a poor quality of life. without headache, with a minimum of 14 days for being considered as successfully fulfilled end-point. These studies showed an imperative clinical use of these PSP toxins, the therapeutics properties of these biotoxins during a local The immediate effect of the toxin, as well as to control a placebo

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effect, EMG recording was performed, 2 minutes after toxin or place- Considering the amazing immediate relaxant effect observed in bo injections. The injection pain scores and pain duration in minutes this study, but also reported for healing of anal fissures [30, 31], the was evaluated by asking the patients to rate their pain in a visual primary outcome and major improvement aimed by the authors in this analog scale (VAS scale) from 1 to 10, being 10 the maximum pain study was to produce a significant drop in patient’s acute pain score. value. Additionally, they were asked to rate the pain felt during the This outcome was achieved for the 83 percentage of responder patients, infiltration, as well as amount of paresthesia in the injected muscles, considering a 30 percentage drop off of the baseline headache pain. The anesthesia or any other discomfort or pain after the infiltration. following outcome measure was the number of days without headache episodes. This was remarkably achieved since the average duration of Apart of oral paraesthesia reported by 12 of the 27 patients the prophylactic effect provided by the Gonyautoxin 2/3 epimers was included in the trial, no other negative symptoms were described or above 8 weeks, meaning an average of 2 months without headache. detected during the follow up period. When a two-week period was Compared with the available treatments for chronic tension-type considered as a base line, 19 of 27 patients (70%) were successfully headache [50-52], this is an excellent prophylactic long lasting effect responders to the infiltration. The patients showed a remarkable effect The effect is superior to that provided by the gold standard based on immediately after the infiltration, confirmed by trapezium EMG tricyclic anti depressive reporting a 30 % efficacy [48]. recording. All patients reported a decrease in pain score at 5 minutes post-injection. The responder above the baseline (two weeks), showed The gonyautoxin infiltration was well tolerated and immediately an average above 8 weeks of headache pain free, all of them without a effective. The beneficial effects lasted for an average over 2 months and second infiltration or the usage of any additional analgesic in some patients for several months. This long lasting anesthetic effect medication. with some patients showing 50 weeks without headache pain was above of the one expected in the study, being this study the first trial testing The pain information recorded was: average headache pain gonyautoxin in treatment of chronic tension-type headache, no data is during the week before infiltration, value of pain scores just before the available to compare such as long lasting pain relief. infiltration and 5 minutes after the infiltration. The physiological effects of this potent voltage sodium channel Follow-up was performed by telephone 48 hours after the blocker may include a wide range of neurophysiological and neuro- treatment for each patient. A visiting follow-up was performed 1 and chemical responses such as release of neurotransmitters or activation of 2 weeks after treatment. The patients were asked to score the overall segmental and heterosegmental antinociceptive system. On the other effect of the treatment and to report any side effects. Both intensity hand, since chronic pain is considered as an expression of neuronal and frequency were scored. All patients were contacted by telephone plasticity and it is generated partly in the periphery and partly in the every two weeks for a long-term follow up for 50 weeks. The EMG CNS, maybe this treatment with gonyautoxin could influence changes recordings showed a dramatic abolition of the trapezius muscle of the central sensitization generated by prolonged nociceptive input activity after toxin injection, confirming its relaxant effect. This effect from the pericranial myofascial tissues. This central sensitization was never observed with placebo. change has been postulated as a physiopathological change in tension- The gonyautoxin infiltration showed an abrupt and effective type headache [53]. decrease in acute pain scores in minutes, been the immediate clinical Achalasia: Intrasphincteric Neosaxitoxin infiltration. Evidence effect of the infiltration. The patient sensation it was undoubtedly for lower esophageal sphincter relaxation demonstrated by the EMG recording of the muscle activity post infiltration. The trapezium activity was practically abolished two Following the successful experience with PSP toxins treatment as minutes after infiltration. EMG recording was in agreement with the smooth muscle relaxant for chronic anal fissure [30, 31, 37], the author immediate pain relief reported by the patients after the infiltration, tested Neosaxitoxin to yield lower esophageal sphincter relaxation in and the very low pain reported after the toxin injection [Figure 2] two severe Achalasia cases [34]. [39]. Achalasia is a gastrointestinal motility disorder characterized by aperistalsis of the thoracic esophagus and failure of the lower esophageal sphincter to relax during swallowing [54]. Clinically is expressed by dysphagia for solids and liquids, regurgitation of undigested food, chest pain and weight loss [55, 56]. The diagnosis is confirmed by radiological evaluation and manometric outcomes. The current methods of achalasia treatment are focused on reducing the lower esophageal sphincter (LES) pressure, thus improving the esophageal emptying. These methods include pharmacological therapy [57-62], mechanical disruption of the LES fibers (either by pneumatic dilatation or by surgical myotomy) and the use of botulin toxin [63-69]. Two patients with long standing history of achalasia were submitted to local intrasphincteric endoscopically infiltrations with Neosaxitoxin, both were diagnosed with upper endoscopies. They showed alimentary remnants in a dilated and atonic esophagus, a tight closed LES, but with no other pathology. The Barium esophagram revealed a typical “bird’s beak” appearance and a dilated and tortuous esophagus. The intraesophageal pressure recording showed an absent esophageal peristalsis, impaired LES relaxation and, in both cases, resting LES Figure 2: Cystoscopy infiltration protocol model. Each white dot represents a pressure was within normal range. site of injection.

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In order to perform the infiltration, a flexible upper gastrointe- Since Neosaxitoxin is a phycotoxin that reversibly blocks the stinal endoscopy was performed while the patients were under voltage-gated sodium channels at the neuronal level and being the conscious sedation. The LES was visualized endoscopically by the most potent of these toxins, it should be a strong pain killer. Indeed, identification of the “sphinteric rosette” at the level of the squamous- Neosaxitoxin has shown greater potency than other STX analogous columnar junction. Through a 6-mm sclerotherapy needle, one and it is also more potent than TTX in vitro and in vivo animal milliliter of sterile saline solution with s dose of 5 8micrograms of studies [74, 82]. The relative potencies of these toxic compounds in Neosaxitoxin was infiltrated into each of the four sphincter quadra- vitro and in vivo experiments have been described: Neosaxitoxin > nts. The procedure was performed in an outpatient basis. STX> TTX [74, 79]. The symptomatic response was evaluated on the basis of a The first pioneer study was conducted as randomized, double- symptom achalasia score according to Echards, which was the sum of blind, placebo-controlled trial, with 10 healthy volunteers [35]. the individual score for three achalasia symptoms: dysphagia, Subcutaneous injections were made in the middle posterior skin of regurgitation and chest pain [64]. Patient 1 started with an achalasia the calf, one leg receiving 50 micrograms of neosaxitoxin and the score of 9 and patient 2 with a score 8. Esophageal manometries were contra-lateral receiving the placebo. The anesthetic effect was performed before and 16 hours after the infiltration, both were evaluated using a standardized human sensory and pain model, TSA treated ambulatory and evaluated every day. Clinical follow up was II Neurosensory Analyser (Medoc Ltd, Minneapolis, MN) and also made using a standardized symptoms scale. Patients were contacted the Von Frey Technique were used to evaluate five parameters: daily by phone for the next 10 days. sensory threshold for warm and cold, pain thresholds for heat and cold and mechanical touch perception threshold. Measurements were Similarly, to anal fissure the immediate sphincter relaxation was made at 0, 1, 3, 6, 9, 12, 16, 24 and 48 hours after the injections. All observed and recorded at 5 minutes after infiltration. The resting thermal thresholds were determined as the average of three lower esophageal sphincter pressure fall down in an average of 40%. assessments performed at 10 second intervals, from a baseline After two hours the symptoms improved significantly, achieving a temperature of 32ºC, and with a rate of change of 1ºC/second. The score of cero at four hours after infiltration. The clinical relaxation upper cut-off limit was 50ºC and the lower 0ºC. Cold and warm effect lasted for eight days. Again, the long lasting effect was observed. detection thresholds were defined as the smallest change from Dysphagia was the first symptom to revert, followed by baseline that the volunteer could perceive, and the volunteer pressed a regurgitation and then chest pain. At the day fourteen after injection, button as soon as the specific sensation was perceived. The heat and the surgical myotomy was performed without difficult. No evidence cold pain detection threshold was the temperature perceived as of periesophageal adhesions was found at the surgical time. Muscle painful, and the volunteers were instructed to react to the first samples were taken at the myotomy site in order to perform a sensation of pain. histological evaluation of inflammation. Biopsy revealed depletion of The touch detection thresholds were determined by mechanical ganglion cells in the myenteric plexus, with no inflammatory stimuli with a series of monofilaments of different strength. Ten hairs infiltration at the muscular level. No side effects were observed were used that covered the range from 0.1 to 100 force grams on a during and after the infiltration. Meaning that the intrasphincteric logarithmic scale. (Touch Test Sensory Evaluators, Stoelting Co, Neosaxitoxin infiltration was effective and safe in treatment of Wood Dale, IL) The touch detection threshold was defined as the Achalasia. This therapeutic approach does not contribute to any least force of mechanical stimulation that produced a sensation of additional risk as was showed during this protocol. touch or pressure. Neosaxitoxin as a local anaesthetic Localized reactions at the injection site (erythema, discoloration, The voltage-gated sodium channels are responsible for the rising hematoma, induration, swelling and blisters) and Neosaxitoxin phase of the action potential in the membranes of neurons [70]. At intoxication symptoms like nausea, headache, ataxia, perioral and least nine distinct voltage-gated sodium channels have been cloned distal limbs paraesthesia were recorded. No adverse reactions to from mammals, being broadly divided by its affinity to Neosaxitoxin were detected. Four volunteers presented small (TTX). The NaV1.1, NaV1.2, NaV1.3 and NaV1.7 are highly TTX haematomas in the infiltration zone, two in the toxin group and two sensitive, whereas NaV1.5, NaV1.8 and NaV1.9 are TTX resistant to in the placebo group at the 24 evaluation hours, both disappeared varying degrees [71]. Many of these channels have specific tissue after two weeks. No other local reactions were observed. None of the distributions, determining distinct excitation properties [72]. volunteers noted or showed any motor disability or discomfort during the follow up period. Two weeks after the injections the Local anaesthetics are compounds that reversibly block the neural volunteers returned to the Clinical Hospital where physicians conduction by occupying enough sodium channels within an axon to evaluated the injection sites for persistent and delayed reactions and interrupt its activity, stopping the depolarization, thus preventing the the volunteers were questioned regarding any abnormality. Two propagation of action potential and neuronal communication [73]. months after the injections, the volunteers were contacted by The clinical use of local anaesthetics such as aminoamides (e.g. telephone and asked about any trouble or incidents that they felt ) and amino esters (e.g. procaine), inhibit sodium channel might be related to the study applications. activity by binding in the channel inner pore, toward the inside from the intracellular side of the cell [70]. The data showed an effective blocking outcome of the evaluated parameters in all patients. Gradually the blocking began to revert, the In the last 25 years, there has been an interest in the local heat pain being the first to return to normal values after 3 hours. Cold anaesthetic activity of biotoxins. They bind to the outer opening of pain was the longest sensation abolished, achieving 24 hours of sodium channels [74]. These agents, including TTX and saxitoxin blockade. The touch detection thresholds presented a significant (STX) analogues, reversibly bind to sodium channels with high reduction that last until 9 hours post injection. This one also showed affinity [75-81]. a progressive return to normal values.

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Neosaxitoxin was undetected, by High Performance Liquid Chro- Neosaxitoxin (10 micrograms) plus bupivacaine (5 milligrams), and matography (HPLC) with on line fluorescence detection method [26], (iv) Neosaxitoxin (10 micrograms) plus epinephrine (1:100.000). The in blood and urine samples collected at 1 and 4 hours after infiltra- volunteers received all 4 formulations, with one month elapsing tion. Nevertheless, Neosaxitoxin showed to be a potent agent capable between the two rounds of experiments. The validated sensory and of producing the anaesthetic effect of outer sodium channel blocking pain paradigm performed by Rodriguez [35] was again used for compounds like TTX and STX, whose effects have long been known, evaluating the effect of the treatments from 0 to 72 hours after the since they were previously tested only in animal models [79, 83-87]. injections, measuring sensory pain and mechanical touch perception The clinical use of Neosaxitoxin showed to be effective and safe threshold. Two rounds of experiments were performed, every as local anaesthetic when injected in the subcutaneous plane. More- participant receiving 2 of the 4 combinations in each round, one on over, at microgram dosage level a clinically sensitive blockage, in a each forearm. Therefore, each participant received the 4 preparations completely reversible manner was observed.The data displayed in this during the trial. The rounds were performed with one month interval. report opened a whole new line of research in acute and chronic pain The combination and the forearm to be injected were defined management with this biotoxin. according to a computer-generated randomization table. The volu- nteer and the physician administrating the solutions were blind to the Development of a new local anesthesic content of the treatment. Essentially, local anesthetics effectively block and relieve pain, Two weeks after the injections the volunteers returned to the but with a relatively short duration of action, limiting its anesthetic Clinical Hospital, where physicians evaluated the injection sites for effectiveness not more than 4 to 6 hours [88]. Therefore, to develop a persistent and delayed reactions. The volunteers were further long-acting pain blocker should improve the management of pain, questioned for any abnormality. Two months after the injections, the but no such agent is yet available for clinical usage. volunteers were contacted by telephone and asked about any trouble Pain relief is a public health issue of such critical relevance as to or incidents that they could associate to the study. constitute an international imperative and fundamental human right The data showed undoubtedly that the duration of the effect [89]. It has been estimated that nearly 80% of patients that undergo a produced by combined treatments was longer than single drugs. In surgical procedure suffer post-operative pain and approximately 20 this study, the authors showed the potentiation of the local anesthesic percentage experience severe pain [90]. Inadequate pain treatment effect of Neosaxitoxin in combination with bupivacaine or epine- results in unnecessary patient suffering, physiological disarrangement, phrine in a randomized double blind clinical trial. The synergistic reduced mobility, delayed ambulation, increased risk to present a effect produced by these drugs was observed in all sensory and pain medical complication and psychological sequels, such as depression, tested. Bupivacaine alone was the shorter blocking agent, followed by anxiety and stress [91]. All of the above disarrangement will extend Neosaxitoxin alone. The Neo-Bupi combination showed longer the hospitalization length and also augment of overall health costs. blocking effect than that produced by Neo-Epi combination, mainly Local anesthesic relieve pain avoiding the systemic side effects in the warm sensation threshold test, suggesting greater efficacy for produced by other analgesics, such as the opioids [89, 93]. Therefore, the Neo-Bupi combination that last over 24 hours in practically all the these types of drugs have been increasingly considered in modern parameters measured. These results agree with data obtained with surgical pain management protocols [88, 94-101]. As a result, it has animal models [85, 87]. been an increased interest for developing a long lasting local The potentiation effect associated with the combination of either anesthesic that can be established by a single injection [88, 94, 95, 97, Neo-Bupi or Neo-Epi may be related to a vasoconstriction produced 98]. by either epinephrine or low doses of bupivacaine, as shown that Until now, Tetrodotoxin, Saxitoxin and Neosaxitoxin have been vasoconstrictors enhance and extend the duration of amide type-local found to yield local anesthesic properties in animal models [79, 83]. anesthesia [102]. One logical explanation for this prolonged effect is Moreover, they also showed that the effect of the outer pore-sodium that epinephrine reduces the drug elimination from the injection site, channel blockers is potentiated when are co-injected with conve- probably by decreasing local blood flow [103]. Vasoconstriction, as ntional local anesthesic or vasoconstrictors [74, 85]. The co-injection reported for conventional local anesthesia, might also reduce the yielded a blockage greater than the sum of the effect of the individual Neosaxitoxin clearance from the action site. Another explanation for drugs [74]. that synergism is that the sodium channel pore is simultaneously blocked on the external and internal sites, by Neosaxitoxin and Since, Neosaxitoxin effect persisted over 24 hours [35] and bupivacaine, respectively, in agreement with the idea of two separates following the idea of developing a long-acting pain blocker, the logic and independent sites on the sodium channel, implying that the way to go was to evaluate the potentiation of the Neosaxitoxin local occupation of one site does not restrict or limit the access to the other anesthesic effect in combination with bupivacaine or epinephrine in a [104]. randomized double blind clinical trial in humans. The precise magnitude of this potentiation has to be tested in So, ten healthy male volunteers aged 18 to 34 were enrolled. The future studies associated to specific pathologies where pain is the inclusion criterion was healthy males with the ability to understand main issue. Nevertheless, the maximal lasting effect showed by either and perform the tests. The following exclusion criteria were drug alone was amplified among 1.5 to 3 times in magnitude when considered: (i) consumption of any oral analgesics at least ten days both drugs were applied together. before the study; (ii) drug abuse history; (iii) any sign of psychiatric disorders at the clinical assessment for recruiting the volunteers. The safety of Neosaxitoxin is again showed in this study; this is Thevolunteers were subcutaneously injected into the left and right the second clinical trial where the anesthesic effect of Neosaxitoxinis forearms with a randomized pair of the following treatments: (i) shown in humans. bupivacaine (5 milligrams); (ii) Neosaxitoxin (10 micrograms); (iii)

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The long acting anesthesia and potentiation subjects are very Caucasian [110, 111]. The prevalence in USA is to 60-70 cases per relevant for current medical practice. A long acting local anesthesic 100,000 [108]. While BPS has a multi-factorial etiology, the most combination would be an important component of a multimodal accepted theory corresponds to an injury or dysfunction of the analgesic management. Multimodal analgesia, in which different glycosaminoglycan layer, which shields the urothelium [112]. This types of analgesics are used in order to block distinct sites of the pain alteration may change the urothelium’ s permeability via abnormal pathway. This is more and more recognized as an effective diffusion of toxic compounds from the urine to the submucosa, management of clinical pain [105]. leading to sensory nerve activation, neurogenic inflammation, pain, The local nerve conduction blocking can be a supplement for and fibrosis, with pain being the most distinctive symptom reported systemic analgesics, such as non-steroidal anti-inflammatory drugs by patients. This injury can be caused by bacterial cystitis, childbirth, and opioids. Additionally, this strategy would allow a reduction of the pelvic surgery, or urological procedures [109, 112]. The report respective doses, decreasing the incidence and severity of adverse analysed now describes the therapy involving local infiltration of reactions. The data showed in these studies supports the idea that Neosaxitoxin in urothelium submucosa to block pain caused by BPS, Neosaxitoxin is a new long-acting pain blocker, with highly potential thus improving patient’s quality of life of patients with this chronic, clinical usage, that will play a crucial role in the management of debilitating condition. clinical pain in order to improve the patients’ quality of life. The authors designed this study to also evaluate the clinical Based on the promising results obtained with the clinical use of efficacy of Neosaxitoxin as a long- lasting pain blocker in the Neosaxitoxin, a randomized, double blind clinical trial comparing treatment of Bladder Pain Syndrome (BPS). Patients with a diagnosis Neosaxitoxin versus Bupivacaine for wound infiltration in patients of BPS received a total dose of 80 micrograms of Neosaxitoxin in undergoing laparoscopic cholecystectomy was performed by 0.9% NaCl, pH 6.5, isosmotic. The infiltration was performed via Rodriguez-Navarro [106]. In this study, the authors follow the cystoscopy, under spinal anesthesia. Questionnaires were applied postoperative course of patients undergoing laparoscopic cholecyste- immediately before and 7, 30, and 90 days after the procedure to ctomy under a standardized general anesthesia plus wound infiltra- measure the patients’ reported pain severity and quality of life. tion with either Neosaxitoxin or bupivacaine. In this pilot study, again all patients successfully responded to The aim was to compare both local blocking pain effects after 12 treatment. Furthermore, the analgesic effect lasted for the entire 90 hours post-surgery. Patients received pre-incisional infiltration of days of follow-up, without the need of a second infiltration, and no laparoscope entry sites with 20 millilitres containing either adverse reactions to Neosaxitoxin were detected. Upon infiltration, Neosaxitoxin total dose of 100 micrograms or Bupivacaine 50 all patients who were previously refractory to conservative, as well as milligrams total dose. The primary outcome measured was the visual more aggressive treatments, showed significant clinical improve- analog pain score at 12 hours post-surgery. The second one included ments. Nevertheless, pain the main symptom of BPS, was blocked. It patient pain scores at rest or allowed to move. Additionally, the extra is important to highlight that the analgesic effect produced by analgesic usage, the functional recovery, and any possible adverse Neosaxitoxin infiltration lasted for the entire 90 days of follow-up in effects were also recorded. The study includes 137 patients, 69 all patients, and this pain blocking effect only required one local randomized to Neosaxitoxin and 68 to Bupivacaine infiltrations. intervention. Therefore, Neosaxitoxin infiltration generates a pain blockage that is locally maintained over a long period of time (above Average pain scores at rest and with movement 12 hours post- days). Furthermore, no patient experienced adverse effects such as surgery were lower in the Neosaxitoxin group compared with the nausea or ataxia during or after this treatment for the three months bupivacaine group (PG 0.01). Additional pain measures and recovery of clinical follow-up. For the first time, the effects of Neosaxitoxin on parameters also favoured Neosaxitoxin. No serious adverse events blocking the neuronal transmission of pain is shown, when is locally occurred, and none adverse events were more frequent in the infiltrated into the bladder submucosa. Neosaxitoxin treated group. Neosaxitoxin shows promising as a long- acting local anesthesic. Future studies will examine dose response, The BPS significantly impacts the quality of life of patients by formulation of combinations, and safety with dose escalation. generating debilitating pain and urinary frequency and urgency that, in some cases, leads to social isolation. For this reason, many women A Long-acting pain blocker affected by BPS have associated psychiatric disorders, such as depression and anxiety [108]. Other medical conditions associated Neosaxitoxin as a long-acting pain-blocker in Bladder Pain with BPS are inflammatory and painful chronic conditions such as Syndrome fibromyalgia, vulvodynia, irritable bowel syndrome, and chronic Bladder Pain Syndrome (BPS) is defined as a “chronic pelvic pain, fatigue syndrome [108, 113, 114]. Authors reported, that after pressure, or discomfort of greater than 6 months duration perceived Neosaxitoxin infiltration, all patients had remission of their to be related to the urinary bladder, accompanied by at least one psychiatric disorders associated with this pathology. Consequently, other urinary symptom, like persistent urge to void or urinary each woman had improved quality of life, which was evidenced by frequency. Confusable diseases as the cause of the symptoms must be the reduction in the Interstitial Cystitis Problem Index (ICPI score). excluded” [107, 108]. The Bladder Pain Syndrome Committee of the Moreover, with this treatment, all psychiatric and pharmacological International Consultation on Incontinence has referred to costs were eliminated. Interstitial Cystitis (IC) as the BPS since 2010 [107, 108]. BPS is not a This pilot study, showed the efficacy and safety of Neosaxitoxin life-threatening illness, but it has recently been acknowledged as a as a long acting pain-blocker for BPS. It was well tolerated by major health issue, which seriously affects patients’ quality of life, and patients, who experienced extended pain relief and associated is often accompanied, by sleep and depressive disorders, anxiety, and beneficial effects over the 90 days of follow-up. recurrent urinary tract infections. Consequently, ordinary daily activities are usually avoided [109]. This syndrome affect both sexes, Until now, Neosaxitoxin has only been clinically applied in the but women are more commonly affected and 90% of patients are Hospital Clínico de la Universidad de Chile in Santiago, where a

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pioneering collaboration between basic science investigators and 15. Negri A P and G J Jones. Bioaccumulation of paralytic shellfish poisoning (PSP) toxins from the cyanobacterium Anabaena circinalis by the freshwater mussel clinicians has demonstrated the therapeutic properties of these Alathyriacondola. Toxicon. 1995; 33:667-678. [Crossref] biotoxins upon a local infiltration intervention, which is both effective 16. Lagos N, H Onodera, A Zagatto, DO Andrinolo, SMFQ Azevedo, Y Oshima, et al. and safe [115]. The first evidence of paralytic shellfish toxins in the freshwater cyanobacteriumCylindrospermopsisraciborskii, isolated from Brazil.Toxicon.1999; Conclusion 37:1359-1373. [Crossref] Paralytic Shellfish Poison Toxins, when are applied locally, have 17. Sant'Anna C L, L R de Carvalho, M F Fiore, M E Silva-Stenico, A S Lorenzi, F R Rios, et al. Highly toxic Microcystisaeruginosa strain, isolated from Sao Paulo- been established to be effective and innocuous in all the clinical trials. Brazil, produce hepatotoxins and paralytic shellfish poison neurotoxins. Neurotox They showed a remarkable muscle relaxant and an amazing local pain Res. 2011; 19:389-402. [Crossref] killer effect. Both properties usually occur and aren’t possible to 18. Pomati F, S Sacchi, C Rossetti, S Giovannardi, H Onodera, Y Oshima, et al. The separate, also are manifested almost instantly. Considering instantly, Freshwater CyanobacteriumPlanktothrix Sp. Fp1: Molecular Identification and as the outcome relaxation and/or anesthesic effect measured and Detection of Paralytic Shellfish Poisoning Toxins. J Phycol. 2000; 36:553-562. [Crossref] recorded in minutes after the injection. Neosaxitoxin, the most potent of all PSP toxins, also showed to be a strong long-lasting local 19. Pereira P, H Onodera, D oAndrinolo, S Franca, F Araújo, N Lagos ,et al. 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37. Torres R, Pizarro L, CsendesA,García C and Lagos N. GTX 2/3 epimers permeate the 62. Martinek JM Siroky, Z Plottova, J Bures, A Hep, J Spicak, et al. Treatment of intestine through a paracellular pathway. J Toxicol Sci. 2007; 32: 241-248. patients with achalasia with botulinum toxin: a multicenter prospective cohort study. [Crossref] Dis Esophagus. 2003; 16:204-209. [Crossref] 38. García C, Rodriguez-Navarro A and Días J.C. Evidence of in-vitro glucuronidation 63. Csendes A, I Braghetto, AHenriquez and C Cortes. Late results of a prospective and enzymatic transformation of paralytic shellfish toxins by healthy human liver randomised study comparing forceful dilatation and oesophagomyotomy in patients microsomes fraction. Toxicon. 2009; 53:206-213. [Crossref] with achalasia. Gut. 1989; 30:299-304. [Crossref] 39. Lattes K, P Venegas, N Lagos, M Lagos, L Pedraza, A J Rodriguez-Navarro, et al. 64. Eckardt VF, C Aignherr and G Bernhard. 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