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Anti-Strychnine Immunoconjugate Reduces the Effects of Strychnine-Induced Toxicity in Mice
Abstract
Strychnine poisoning induces seizures that result in loss of control of airway muscles, leading to asphyxiation and subsequent death. Current treatment options are limited, requiring hands-on medical care and isolation to low-stimulus environments. Anticonvulsants and muscle relaxants have shown limited success in cases of severe toxicity. Furthermore, nonfatal strychnine poisoning is likely to result in long-term muscular and cognitive damage. Due to its potency, accessibility, and lack of effective antidotes, strychnine poses a unique threat for mass casualty incidents. As a first step toward developing an anti-strychnine immunotherapy to reduce or prevent strychnine-induced seizures, a strychnine vaccine was synthesized using subunit keyhole limpet hemocyanin. Mice were vaccinated with the strychnine immunoconjugate and then given a 0.75 mg/kg IP challenge of strychnine and observed for seizures for 30 min. Vaccination reduced strychnine-induced events, and serum strychnine levels were increased while brain strychnine levels were decreased in vaccinated animals compared to the control. These data demonstrate that strychnine-specific antibodies can block the seizure-inducing effects of strychnine and could be used to develop a therapeutic for strychnine poisoning.
Centruroides huichol scorpion venom is lethal to mammals. Analysis of the venom allowed the characterization of four lethal toxins named Chui2, Chui3, Chui4, and Chui5. scFv 10FG2 recognized well all toxins except Chui5 toxin, therefore a partial neutralization of the venom was observed. Thus, scFv 10FG2 was subjected to three processes of directed evolution and phage display against Chui5 toxin until obtaining scFv HV. Interaction kinetic constants of these scFvs with the toxins were determined by surface plasmon resonance (SPR) as well as thermodynamic parameters of scFv variants bound to Chui5. In silico models allowed to analyze the molecular interactions that favor the increase in affinity. In a rescue trial, scFv HV protected 100% of the mice injected with three lethal doses 50 (LD50) of venom. Moreover, in mix-type neutralization assays, a combination of scFvs HV and 10FG2 protected 100% of mice injected with 5 LD50 of venom with moderate signs of intoxication. The ability of scFv HV to neutralize different toxins is a significant achievement, considering the diversity of the species of Mexican venomous scorpions, so this scFv is a candidate to be part of a recombinant anti-venom against scorpion stings in Mexico.
Background
Intramuscular antitoxin is recommended in tetanus treatment, but there are few data comparing human and equine preparations. Tetanus toxin acts within the CNS, where there is limited penetration of peripherally administered antitoxin; thus, intrathecal antitoxin administration might improve clinical outcomes compared with intramuscular injection.
Methods
In a 2 × 2 factorial trial, all patients aged 16 years or older with a clinical diagnosis of generalised tetanus admitted to the intensive care unit of the Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam, were eligible for study entry. Participants were randomly assigned first to 3000 IU human or 21 000 U equine intramuscular antitoxin, then to either 500 IU intrathecal human antitoxin or sham procedure. Interventions were delivered by independent clinicians, with attending clinicians and study staff masked to treatment allocations. The primary outcome was requirement for mechanical ventilation. The analysis was done in the intention-to-treat population. The study is registered at ClinicalTrials.gov, NCT02999815; recruitment is completed.
Findings
272 adults were randomly assigned to interventions between Jan 8, 2017, and Sept 29, 2019, and followed up until May, 2020. In the intrathecal allocation, 136 individuals were randomly assigned to sham procedure and 136 to antitoxin; in the intramuscular allocation, 109 individuals were randomly assigned to equine antitoxin and 109 to human antitoxin. 54 patients received antitoxin at a previous hospital, excluding them from the intramuscular antitoxin groups. Mechanical ventilation was given to 56 (43%) of 130 patients allocated to intrathecal antitoxin and 65 (50%) of 131 allocated to sham procedure (relative risk [RR] 0·87, 95% CI 0·66–1·13; p=0·29). For the intramuscular allocation, 48 (45%) of 107 patients allocated to human antitoxin received mechanical ventilation compared with 48 (44%) of 108 patients allocated to equine antitoxin (RR 1·01, 95% CI 0·75–1·36, p=0·95). No clinically relevant difference in adverse events was reported. 22 (16%) of 136 individuals allocated to the intrathecal group and 22 (11%) of 136 allocated to the sham procedure experienced adverse events related or possibly related to the intervention. 16 (15%) of 108 individuals allocated to equine intramuscular antitoxin and 17 (16%) of 109 allocated to human antitoxin experienced adverse events related or possibly related to the intervention. There were no intervention-related deaths.
Interpretation
We found no advantage of intramuscular human antitoxin over intramuscular equine antitoxin in tetanus treatment. Intrathecal antitoxin administration was safe, but did not provide overall benefit in addition to intramuscular antitoxin administration.
Funding
The Wellcome Trust.
Background: Digoxin poisoning leads to the development of cardiac and noncardiac complications. Digoxin immune fab is a safe and effective antidote, but clinical trials have not been performed in this regard, and most of the evidence is based on prospective studies. Understanding the toxicoepidemiology pattern of digoxin poisoning could provide valuable context for better managing its acute poisoning.
Objectives: This study aimed to assess the toxicoepidemiology pattern of acute digoxin poisoning through a 5-year assessment in Iran.
Methodology: In this observational study, the records of 97 patients who were referred with acute digoxin poisoning between 2010 and 2015 were evaluated. Demographic characteristics, past medical history, drug history, chief complaints, vital signs, paraclinical findings, digoxin immune fab administration, and clinical outcomes recorded.
Results: The mean age of patients was 34.02 ± 17.87 years old. About 24.7% of patients had underlying diseases, and among them, heart failure was the most prevalent disease (29.2%) 42.3% of patients needed intensive care unit (ICU) admission. The mean duration of ICU stay was 4.00 ± 2.29 days. Digoxin immune fab was administered for 4.1% of patients, and an average of 6.2 ± 2.2 vials were used for them. All patients survived, and no mortality was reported.
Conclusions: Digoxin immune fab administration did not alter the mortality rate. Hence, it can be concluded digoxin immune fab is not appropriate in acute poisoning, but it may be considered in chronic poisoning. Furthermore, acute digoxin poisoning is more common in Iran, and it responds appropriately to conventional treatment.
The prevalence and societal impact of opioid use disorder (OUD) is an acknowledged public health crisis that is further aggravated by the current pandemic. One of the devastating consequences of OUD is opioid overdose deaths. While multiple medications are now available to treat OUD, given the prevalence and societal burden, additional well-tolerated and effective therapies are still needed. To this point, we have developed chimeric monoclonal antibodies (mAb) that will specifically complex with fentanyl and its analogs in the periphery, thereby preventing them from reaching the central nervous system. Additionally, mAb-based passive immunotherapy offers a high degree of specificity to drugs of abuse and does not interfere with an individual’s ability to use any of the medications used to treat OUD. We hypothesized that sequestering fentanyl and its analogs in the periphery will mitigate their negative effects on the brain and peripheral organs. This study is the first report of chimeric mAb against fentanyl and its analogs. We have discovered, engineered the chimeric versions, and identified the selectivity of these antibodies, through in vitro characterization and in vivo animal challenge studies. Two mAb candidates with very high (0.1–1.3 nM) binding affinities to fentanyl and its analogs were found to be effective in engaging fentanyl in the periphery and blocking its effects in challenged animals. Results presented in this work constitute a major contribution in the field of novel therapeutics targeting OUD.
A fundamental issue of the characterization of single-chain variable fragments (scFvs), capable of neutralizing scorpion toxins, is their cross-neutralizing ability. This aspect is very important in Mexico because all scorpions dangerous to humans belong to the Centruroides genus, where toxin sequences show high identity. Among toxin-neutralizing antibodies that were generated in a previous study, scFv 10FG2 showed a broad cross-reactivity against several Centruroides toxins, while the one of scFv LR is more limited. Both neutralizing scFvs recognize independent epitopes of the toxins. In the present work, the neutralization capacity of these two scFvs against two medically important toxins of the venom of Centruroides sculpturatus Ewing was evaluated. The results showed that these toxins are recognized by both scFvs with affinities between 1.8 × 10⁻⁹ and 6.1 × 10⁻¹¹ M. For this reason, their ability to neutralize the venom was evaluated in mice, where scFv 10FG2 showed a better protective capacity. A combination of both scFvs at a molar ratio of 1:5:5 (toxins: scFv 10FG2: scFv LR) neutralized the venom without the appearance of any signs of intoxication. These results indicate a complementary activity of these two scFvs during venom neutralization.
The interest in substances that stimulate respiration has waxed and waned throughout the years, intensifying following the introduction of a new class of drugs that causes respiratory depression, and diminishing when antidotes or better drug alternatives are found. Examples include the opioids––deaths increasing during overprescribing, diminishing with wider availability of the opioid receptor antagonist naloxone, increasing again during COVID-19; the barbiturates––until largely supplanted by the benzodiazepines; propofol; and other central nervous system depressants. Unfortunately, two new troubling phenomena force a reconsideration of the status-quo: (1) overdoses due to highly potent opioids such as fentanyl, and even more-potent licit and illicit fentanyl analogs, and (2) overdose due to polysubstance use (the combination of an opioid plus one or more non-opioid drug, such as a benzodiazepine, sedating antidepressant, skeletal muscle relaxant, or various other agents). Since these now represent the majority of cases, new solutions are again needed. An interest in respiratory stimulants has been revived. This interest can be informed by a short review of the history of this interesting class of medications. We present a short history of the trajectory of advances toward more selective and safer respiratory stimulants.
Several species of dangerous snakes are found in Iran and, according to the Emergency Response Center of Iran from 2002 to 2011, 53,787 Iranians have suffered from snakebite. Although the mortalities caused by snakebite are very low, snakebite-related amputations are still a major concern. Currently, anti-venom polyclonal antibodies derived from animals, such as horses are used to treat snakebites; however, in some cases they can cause anaphylactic shock and serum sickness. In line with this premise, generation of recombinant anti-venom antibodies can be considered as an alternative strategy. Single-chain fragment variable (scFv) antibodies offer several advantages compared to the whole antibodies, including ease of production, high affinity and specificity. In the present study, scFv antibodies were selected against the venom of the most poisonous snakes in Iran using phage display technology. Phage particles harboring anti-venom specific scFv were separated and scFv antibodies were produced in bacteria. In-vitro assay showed that polyclonal scFvs specifically bind to the venom. Furthermore, in-vivo experiment in mice BALB/c indicated effective toxin neutralization using 20 µg of polyclonal scFv. Our study indicates the neutralizing capacity of anti-venom polyclonal scFvs, although further neutralization assays are needed to confirm their effectiveness.
This work describes the neuropharmacological (sedative, anxiolytic, antidepressant, and anticonvulsant) actions of Gardenin A (GA) (0.1–25 mg/kg p.o.), a flavonoid found in medicinal plants. The sedative effects of GA were assessed with the pentobarbital‐induced sleep test. The anxiolytic actions of GA were evaluated with the elevated plus‐maze, the light–dark box test, the exploratory cylinder assay, and the open field test. Motor coordination was evaluated with the rotarod test and the open field test. The antidepressant‐like actions of GA were evaluated with the tail suspension test and forced swimming test. The mechanisms of the anxiolytic‐like and antidepressant‐like effects of GA were assessed using inhibitors of neurotransmission pathways. The anticonvulsant activity of GA was evaluated with the strychnine‐induced seizure test. The sedative effects of GA were evident only at a dose of 25 mg/kg, which increased the duration of sleep but did not alter sleep onset. GA showed anxiolytic‐like actions with activity comparable to that of clonazepam in all experimental tests. The GABAA receptor antagonist bicuculline reversed the anxiolytic‐like effects of GA. Furthermore, GA showed significant antidepressant‐like actions in both models with activity comparable to that of fluoxetine. Yohimbine, an α2‐adrenoceptor blocker, inhibited the antidepressant‐like actions of GA. In addition, GA (1–10 mg/kg) did not affect locomotor coordination in mice and delayed the onset of convulsions. These findings suggest that GA induces anxiolytic‐like effects and has anticonvulsant actions by the possible involvement of the GABAergic system. The antidepressant‐like actions of GA may be mediated by noradrenergic neurotransmission.
Inhalation of ricin toxin (RT), a Category B biothreat agent, provokes an acute respiratory distress syndrome marked by pro-inflammatory cytokine and chemokine production, neutrophilic exudate, and pulmonary edema. The severity of RT exposure is attributed to the tropism of the toxin’s B subunit (RTB) for alveolar macrophages and airway epithelial cells, coupled with the extraordinarily potent ribosome-inactivating properties of the toxin’s enzymatic subunit (RTA). While there are currently no vaccines or treatments approved to prevent RT intoxication, we recently described a humanized anti-RTA IgG1 MAb, huPB10, that was able to rescue non-human primates (NHPs) from lethal dose RT aerosol challenge if administered by intravenous (IV) infusion within hours of toxin exposure. We have now engineered an extended serum half-life variant of that MAb, huPB10-LS, and evaluated it as a pre-exposure prophylactic. Five Rhesus macaques that received a single intravenous infusion (25 mg/kg) of huPB10-LS survived a lethal dose aerosol RT challenge 28 days later, whereas three control animals succumbed to RT intoxication within 48 h. The huPB10-LS treated animals remained clinically normal in the hours and days following toxin insult, suggesting that pre-existing antibody levels were sufficient to neutralize RT locally. Moreover, pro-inflammatory markers in sera and BAL fluids collected 24 h following RT challenge were significantly dampened in huPB10-LS treated animals, as compared to controls. Finally, we found that all five surviving animals, within days after RT exposure, had anti-RT serum IgG titers against epitopes other than huPB10-LS, indicative of active immunization by residual RT and/or RT-immune complexes.
Opioid abuse in the United States has been declared a national crisis and is exacerbated by an inexpensive, readily available and illicit supply of synthetic opioids. Specifically, fentanyl and related analogues such as carfentanil pose a significant danger to opioid users due to their high potency and rapid acting depression of respiration. In recent years these synthetic opioids have become the number one cause of drug-related deaths. In our research efforts to combat the public health threat posed by synthetic opioids, we have developed monoclonal antibodies (mAbs) against fentanyl class drugs. The mAbs were generated in hybridomas derived from mice vaccinated with a fentanyl conjugate vaccine. Guided by a surface plasmon resonance (SPR) binding assay, we selected six hybridomas that produced mAbs with 10-11 M binding affinity for fentanyl, yet broad cross-reactivity with related fentanyl analogues. In mouse antinociception models, our lead mAb (6A4) could blunt the effects of both fentanyl and carfentanil in a dose-responsive manner. Additionally, mice pre-treated with 6A4 displayed enhanced survival when subjected to fentanyl above LD50 doses. Pharmacokinetic analysis revealed that the antibody sequesters large amounts of these drugs in the blood thus reducing drug biodistribution to the brain and other tissue. Lastly, the 6A4 mAb could effectively reverse fentanyl/carfentanil-induced antinociception comparable to the opioid antagonist naloxone, the standard of care drug for treating opioid overdose. While naloxone is known for its short half-life, we found the half-life of 6A4 to be approximately six days in mice, thus monoclonal antibodies could theoretically be useful in preventing re-narcotization events in which opioid intoxication recurs following quick metabolism of naloxone. Our results as a whole demonstrate that monoclonal antibodies could be a desirable treatment modality for synthetic opioid overdose and possibly opioid use disorder.
Concern about use of anthrax as a bioweapon prompted development of novel anthrax antitoxins for treatment. Clinical guidelines for the treatment of anthrax recommend antitoxin therapy in combination with intravenous antimicrobials; however, a large-scale or mass anthrax incident may exceed antitoxin availability and create a need for judicious antitoxin use. We conducted a systematic review of antitoxin treatment of inhalation anthrax in humans and experimental animals to inform antitoxin recommendations during a large-scale or mass anthrax incident. A comprehensive search of 11 databases and the FDA website was conducted to identify relevant animal studies and human reports: 28 animal studies and 3 human cases were identified. Antitoxin monotherapy at or shortly after symptom onset demonstrates increased survival compared to no treatment in animals. With early treatment, survival did not differ between antimicrobial monotherapy and antimicrobial-antitoxin therapy in nonhuman primates and rabbits. With delayed treatment, antitoxin-antimicrobial treatment increased rabbit survival. Among human cases, addition of antitoxin to combination antimicrobial treatment was associated with survival in 2 of the 3 cases treated. Despite the paucity of human data, limited animal data suggest that adjunctive antitoxin therapy may improve survival. Delayed treatment studies suggest improved survival with combined antitoxin-antimicrobial therapy, although a survival difference compared with antimicrobial therapy alone was not demonstrated statistically. In a mass anthrax incident with limited antitoxin supplies, antitoxin treatment of individuals who have not demonstrated a clinical benefit from antimicrobials, or those who present with more severe illness, may be warranted. Additional pathophysiology studies are needed, and a point-of-care assay correlating toxin levels with clinical status may provide important information to guide antitoxin use during a large-scale anthrax incident.
ABSTRACT This first-in-human study examined the safety and pharmacokinetics of ch-mAb7F9, an anti-methamphetamine monoclonal antibody, in healthy volunteers. Single, escalating doses of ch-mAb7F9 over the range of 0.2 to 20 mg/kg were administered to 42 subjects who were followed for 147 days. Safety was measured by physical examinations, adverse events, vital signs, electrocardiograms, and clinical laboratory testing. Serum ch-mAb7F9 concentration and immunogenicity analyses were performed. There were no serious adverse reactions or discontinuations from the study due to adverse events. No trends emerged in the frequency, relatedness, or severity of adverse events with increased dose or between active and placebo treated subjects. Ch-mAb7F9 displayed expected IgG pharmacokinetic parameters, including a half-life of 17-19 days in the three highest dose groups and volume of distribution of 5-6 L, suggesting the antibody is confined primarily to the vascular compartment. Four (12.5%) of the 32 subjects receiving ch-mAb7F9 were confirmed to have developed a human anti-chimeric antibody response by the end of the study; however, this response did not appear to be dose related. Overall, no apparent safety or tolerability concerns were identified; a maximum tolerated dose was not reached in this Phase 1 study. Ch-mAb7F9 therefore appears safe for human administration.
Context:
Digoxin-specific antibody fragments (digoxin-Fab) are widely regarded as a safe and effective treatment for the management of acute and chronic digoxin poisoning. Calculated equimolar doses of digoxin-Fab are high, very expensive, and infrequently used.
Objective:
To review the pharmacology, efficacy, effectiveness, indications, safety and the dosage of digoxin-specific antibody fragments.
Methods:
Pubmed, Embase, Medline and Cochrane were searched from 1946 to May 2013 using the terms digoxin, digoxin-specific Fab, and digoxin antibody. Pharmacology and kinetics of digoxin and digoxin-Fab. Digoxin acts via inhibition of Na⁺/K⁺ ATPase. It has a narrow therapeutic index. Digoxin has 60-80% bioavailability, a mean plasma half-life of 40 h and a volume of distribution (Vd) of 5-10 L/kg and low protein binding (20%). A 40-mg vial of digoxin-Fab (DigiFab) binds 0.5 mg digoxin. Digoxin-Fab has a mean plasma half-life of 19-30 h and a Vd of 0.4 L/kg. The half-lives of both digoxin and digoxin-Fab are prolonged in renal failure to over 100 h. Efficacy and effectiveness of digoxin-Fab. There were no randomised clinical trials examining the use of digoxin-Fab for acute or chronic digoxin poisonings. Ten case series with a total of 2,080 patients have reported on the use of digoxin-Fab in digoxin poisoning. In three large case series of 430 acute and 1308 chronic poisonings, response rates to digoxin-Fab vary from 80-90% to 50%. The time for reversal of digoxin toxicity is reported to be 30-45 min. Studies with pharmacokinetic data showed that free digoxin concentration fell to almost zero within a few minutes following the administration of digoxin-Fab. Digoxin-Fab was used more frequently in acute than chronic digoxin poisoning with a higher reported success rate when used in acute overdose. It is sometimes recommended to use full neutralisation doses (based on serum concentration × Vd or ingested dose). It has also been proposed to use half this dose. Indications for digoxin-Fab. Patients who have life-threatening tachy-bradyarrhythmias, hyperkalaemia (> 6 mmol/L) or haemodynamic instability with an elevated digoxin concentration (> 2 μg/L or 2.6 nmol/L). The lowest effective digoxin-Fab dosing regimen has not been established. Safety of digoxin-Fab. Adverse events such as exacerbation of heart failure, increased ventricular rate and hypokalaemia are uncommon (< 10%). Recrudescence of digoxin toxicity and allergic reactions are infrequent. Digoxin-Fab dosing in acute poisoning. Digoxin load based on ingested dose will generally overestimate digoxin-Fab doses as bioavailability is 60-80%, and further reduced by vomiting and activated charcoal. Digoxin load based on concentration also will be overestimated when the concentration is taken before distribution is complete (around 6 h). Much smaller doses of digoxin-Fab can eliminate the digoxin in the central compartment (Vd ≈ 55 L). In imminent cardiac arrest, it may be justified to give a full neutralising dose. Otherwise, based on pharmacokinetic modelling, it is recommended to give 80 mg bolus digoxin-Fab, repeated as required according to clinical parameters because the onset of clinical response is usually rapid. Most patients would be expected to require a total of less than half of the calculated neutralising dose using this strategy. Digoxin-Fab dosing in chronic poisoning. Even if digoxin load is estimated following distribution (> 6 h), excessive neutralisation doses may still be calculated because of variation in Vd due to equations failing to account for lean body weight, age and renal failure. In practice, it is suggested to give 40 mg (1 vial) digoxin-Fab at a time and repeat after 60 min if patient is still symptomatic, sooner if patient is clinically unstable. In general, 40-120 mg (1-3 vials) should be sufficient.
Conclusions:
Digoxin-Fab is safe and indicated in all patients with life-threatening arrhythmias and an elevated digoxin concentration. However, calculated full neutralising doses of digoxin-Fab are expensive and may not be required. In acute poisoning, a small bolus of 80 mg, repeat if necessary, titrated against clinical effect, is likely to achieve equivalent benefits with much lower total doses. With chronic poisoning, it may be simplest to give 40 mg (1 vial) digoxin-Fab at a time and repeat after 60 min if there is no response.
Three haptens have been synthesized with linkers for attachment to carrier macromolecules at either the piperidino-nitrogen or via an introduced 3-amino group. Two of the haptens, with a 2-oxopropyl functionality at either C6, or at both the C3 and C6 positions on the 4,5-epoxymorphinan framework, as well as the third hapten (DiAmHap) with diamido moieties at both the C3 and C6 positions, should be much more stable in solution, or in vivo in a vaccine, than a hapten with an ester in one of those positions, as found in many heroin-based haptens. A “classical” opioid synthetic scheme enabled the formation of a 3-amino-4,5-epoxymorphinan which could not be obtained using palladium chemistry. Our vaccines are aimed at the reduction of the abuse of heroin and, as well, at the reduction of the effects of its predominant metabolites, 6-acteylmorphine and morphine. One of the haptens, DiAmHap, has given interesting results in a heroin vaccine and is clearly more suited for the purpose than the other two haptens.
We evaluated the immunogenicity, efficacy, and safety of succinylnorcocaine conjugated to cholera toxin B protein as a vaccine for cocaine dependence.
This 6-site, 24 week Phase III randomized double-blind placebo-controlled trial assessed efficacy during weeks 8 to 16. We measured urine cocaine metabolites thrice weekly as the main outcome.
The 300 subjects (76% male, 72% African-American, mean age 46 years) had smoked cocaine on average for 13 days monthly at baseline. We hypothesized that retention might be better and positive urines lower for subjects with anti-cocaine IgG levels of ≥42μg/mL (high IgG), which was attained by 67% of the 130 vaccine subjects receiving five vaccinations. Almost 3-times fewer high than low IgG subjects dropped out (7% vs 20%). Although for the full 16 weeks cocaine positive urine rates showed no significant difference between the three groups (placebo, high, low IgG), after week 8, more vaccinated than placebo subjects attained abstinence for at least two weeks of the trial (24% vs 18%), and the high IgG group had the most cocaine-free urines for the last 2 weeks of treatment (OR=3.02), but neither were significant. Injection site reactions of induration and tenderness differed between placebo and active vaccine, and the 29 serious adverse events did not lead to treatment related withdrawals, or deaths.
The vaccine was safe, but it only partially replicated the efficacy found in the previous study based on retention and attaining abstinence.
Tetramethylenedisulfotetramine (tetramine; TETS) is a potent convulsant poison that is considered to be a chemical threat agent. To provide a basis for the investigation of antidotes for TETS-induced seizures, we characterized the convulsant activity of TETS in mice and rats when administered by the intraperitoneal, intravenous, oral, and intraventricular routes as a single acute dose and with repeated sublethal doses. In mice, parenteral and oral TETS caused immobility, myoclonic body jerks, clonic seizures of the forelimbs and/or hindlimbs, tonic seizures, and death. The CD₅₀ values for clonic and tonic seizures after oral administration were 0.11 and 0.22 mg/kg, respectively. Intraventricular administration of TETS (5-100 μg) in rats also caused clonic-tonic seizures and death. In mice, repeated sublethal doses of TETS at intervals of 2, 24, and 48 h failed to result in the development of persistent enhanced seizure responsivity ("kindling") as was observed with repeated pentylenetetrazol treatment. In mice, sublethal doses of TETS that produced clonic seizures did not cause observable structural brain damage as assessed with routine histology and Fluoro-Jade B staining 7 days after treatment. However, 1 to 3 days after a single convulsant dose of TETS the expression of glial fibrillary acidic protein, an astrocyte marker, and ionized calcium binding adaptor molecule 1, a microglia marker, were markedly increased in cortex and hippocampus. Although TETS doses that are compatible with survival are not associated with overt evidence of cellular injury or neurodegeneration, there is transient reactive astrocytosis and microglial activation, indicating that brain inflammatory responses are provoked.
Nonketotic hyperglycinemia (NKH) is an inborn error of amino acid metabolism resulting from a defect in the glycine cleavage enzyme system. It is characterized biochemically by elevated concentrations of glycine in plasma, spinal fluid, and urine and clinically by respiratory failure, muscular hypotonia, myoclonic seizures, feeding difficulties, and extreme lethargy. The outcome is poor. Various methods of treatment, including those with oral sodium benzoate and strychnine combined with repeated exchange transfusions, were unsuccessful for sustained improvement. In this report we describe an infant girl with a severe form of nonketotic hyperglycinemia diagnosed at three days of age and treated intravenously with sodium benzoate and strychnine sulfate.
Vaccination against nicotine is being studied as a potential treatment for nicotine dependence. Some of the limitations of vaccination, such as variability in antibody titer and affinity, might be overcome by instead using passive immunization with nicotine-specific monoclonal antibodies. The effects of antibodies on nicotine distribution to brain were studied using nicotine-specific monoclonal antibodies (NICmAbs) with K(d) values ranging from 60 to 250 nM and a high-affinity polyclonal rabbit antiserum (K(d) = 1.6 nM). Pretreatment with NICmAbs substantially increased the binding of nicotine in serum after a single nicotine dose, reduced the unbound nicotine concentration in serum, and reduced the distribution of nicotine to brain. Efficacy was directly related to antibody affinity for nicotine. Efficacy of the highest affinity NICmAb, NICmAb311, was dose-related, with the highest dose reducing nicotine distribution to brain by 78%. NICmAb311 decreased nicotine clearance by 90% and prolonged the terminal half-life of nicotine by 120%. At equivalent doses, NICmAb311 was less effective than the higher affinity rabbit antiserum but comparable efficacy could be achieved by increasing the NICmAb311 dose. These data suggest that passive immunization with nicotine-specific monoclonal antibodies substantially alters nicotine pharmacokinetics in a manner similar to that previously reported for vaccination against nicotine. Antibody efficacy is a function of both dose and affinity for nicotine.
Chronic or excessive (+)-methamphetamine (METH) use often leads to addiction and toxicity to critical organs like the brain. With medical treatment as a goal, a novel single-chain variable fragment (scFv) against METH was engineered from anti-METH monoclonal antibody mAb6H4 (IgG, kappa light chain, K(d) = 11 nM) and found to have similar ligand affinity (K(d) = 10 nM) and specificity as mAb6H4. The anti-METH scFv (scFv6H4) was cloned, expressed in yeast, purified, and formulated as a naturally occurring mixture of monomer ( approximately 75%) and dimer ( approximately 25%). To test the in vivo efficacy of the scFv6H4, male Sprague-Dawley rats (n = 5) were implanted with 3-day s.c. osmotic pumps delivering 3.2 mg/kg/day METH. After reaching steady-state METH concentrations, an i.v. dose of scFv6H4 (36.5 mg/kg, equimolar to the METH body burden) was administered along with a [(3)H]scFv6H4 tracer. Serum pharmacokinetic analysis of METH and [(3)H]scFv6H4 showed that the scFv6H4 caused an immediate 65-fold increase in the METH concentrations and a 12-fold increase in the serum METH area under the concentration-time curve from 0 to 480 min after scFv6H4 administration. The scFv6H4 monomer was quickly cleared or converted to multivalent forms with an apparent t(1/2lambdaz) of 5.8 min. In contrast, the larger scFv6H4 multivalent forms (dimers, trimers, etc.) showed a much longer t(1/2lambdaz) (228 min), and the significantly increased METH serum molar concentrations correlated directly with scFv6H4 serum molar concentrations. Considered together, these data suggested that the scFv6H4 multimers (and not the monomer) were responsible for the prolonged redistribution of METH into the serum.
Before 1900, diphtheria was a deadly infection in Canadian children. Clinical trials in Europe demonstrated that “antitoxin” decreased mortality of the disease. Canadian physicians first utilized the new medication in 1894 and found it most efficacious when administered early in the illness. Two factors hindered the widespread application of the new medication. Imported antitoxin was expensive, and physicians frequently purchased it themselves rather than let their patients expire. In 1916, Connaught Laboratories in Toronto began domestic production of antitoxin, and provincial governments eventually provided access to the life-saving medication for all citizens.
The vast distances between families and physicians in rural districts also made it impossible to access the antitoxin before sick children were beyond hope. During the 1920s, a public health program began to actually prevent diphtheria. Ten years of experience in many regions demonstrated the efficacy of immunization as mortality from diphtheria declined to almost zero.
The incidence of fatal drug overdoses in the United States is an alarming public health threat that has been exacerbated by the COVID-19 pandemic, resulting in over 100,000 deaths between April 2020 and April 2021. A significant portion of this is attributable to widespread access to fentanyl and other synthetic opioids, alone or in combination with heroin or psychostimulants such as cocaine or methamphetamine. Monoclonal antibodies (mAb) offer prophylactic and therapeutic interventions against opioid overdose by binding opioids in serum, reducing distribution of drug to the brain and other organs. Here, we investigated the efficacy of a lead anti-fentanyl mAb, clone HY6-F9, in reversal and prevention of fentanyl-induced toxicity compared to the opioid receptor antagonist naloxone (NLX) in rats. In post-exposure models, rats were challenged with fentanyl, followed by HY6-F9, NLX, or both. HY6-F9 reversed fentanyl-induced antinociception, respiratory depression, and bradycardia, and rats retained protection against additional challenges for at least 1 week. Although intravenous NLX reversed fentanyl-induced respiratory depression more rapidly than mAb alone, kinetics of reversal by intravenous mAb were similar to subcutaneous NLX. Co-administration of mAb and NLX provided greater protection than individual treatments against high doses of fentanyl. Prophylactic administration of mAb reduced the ED50 of NLX approximately 2-fold against 2.25 mg/kg fentanyl. Finally, mAb sequestered fentanyl and its metabolite norfentanyl in serum, and reduced brain concentrations of fentanyl. These results support the translation of mAb as medical interventions alone or in combination with NLX to prevent and reverse fentanyl-related overdose. Significance Statement Fentanyl-related overdoses have increased dramatically in the US and worldwide. Currently, approved pharmacotherapies for treatment of opioid use disorder and reversal of overdose are not sufficient to curb the incidence of opioid-related deaths. Additionally, fentanyl and its potent analogs present a potential risk from use in deliberate poisoning or chemical attacks. This study demonstrates the use of mAb as a countermeasure to fentanyl-induced toxicity in pre- and post-exposure scenarios, supporting their use in combination with the opioid antagonist NLX.
Introduction
For acute digoxin poisoning, it has been recommended to give bolus doses of 10–20 vials or potentially larger than needed doses calculated from dose ingested or the measured concentration. However, a recent revision of internal Poisons Information Centre guidelines prompted a change of our recommendations, specifically instead of large boluses, to use titrating repeated low doses of digoxin antibodies(Digoxin-Fab) based on bedside assessment of cardiac toxicity.
Methods
This is a prospective observational study of patients with acute digoxin poisoning identified through two Poisons Information Centres and three toxicology units. Patient demographics, signs and symptoms of digoxin toxicity, doses and response to Digoxin-Fab, free and bound serum digoxin concentrations. Outcomes were recorded and analysed.
Results
From September 2013 to September 2020, 23 patients with 25 presentations (median age 56 years, females 56%) were recruited. Median dose ingested was 13 mg(IQR: 9.5–25). Median heart rate (HR) was 41 beats/min before treatment. Initial median digoxin and potassium concentrations were 14.5 nmol/L (IQR: 10.9–20) [11.2 µg/L(IQR: 8.4–15.4)] and 5 mmol/L (IQR: 4.5–5.4 mmol/L), respectively. Gastrointestinal symptoms and acute kidney injury were present in 22 patients (88%) and 5 patients (20%), respectively. Four patients received an initial bolus dose of Digoxin-Fab of 5–20 vials. Twenty-one patients received repeated titrated doses (1–2 vials) of Digoxin-Fab and the median total dose was 4 vials (IQR: 2–7.5). Median maximal change in HR post-Digoxin-Fab was 19 beats/min. The median potassium concentration decrease post-Digoxin-Fab was 0.3 mmol/L. Total dose used in the titration group was 25% and 35% of the predicted doses based on the amount of digoxin ingested or measured serum concentration, respectively. Twelve had free digoxin concentrations measured. Free digoxin concentrations dropped to almost zero after any dose of Digoxin-Fab. Ten patients had a rebound of digoxin >2.6 nmol/L (2 µg/L). There were no deaths from acute digoxin toxicity.
Conclusions
The new practice of using small, titrated doses of Digoxin-Fab led to a considerable reduction in total usage and major savings. The clinical response to titrated doses was safe and acceptable in acute digoxin poisoning.
Monoclonal antibodies (mAb) and vaccines have been proposed as medical countermeasures to treat opioid use disorder (OUD) and prevent opioid overdose. In contrast to current pharmacotherapies (e.g., methadone, buprenorphine, naltrexone, and naloxone) for OUD and overdose, which target brain opioid receptors, mAb and vaccine-generated polyclonal antibodies sequester the target opioid in the serum and reduce drug distribution to the brain. Further, mAb offer several potential clinical benefits over approved medications, such as longer serum half-life, high selectivity, reduced side effects, and low abuse liability. Using magnetic enrichment to isolate opioid-specific B cell lymphocytes prior to fusion with myeloma partners, this study identified a series of murine hybridoma cell lines expressing mAb with high affinity for opioids of clinical interest, including oxycodone, heroin and its active metabolites, and fentanyl. In mice, passive immunization with lead mAb against oxycodone, heroin, and fentanyl reduced drug-induced antinociception and the distribution of the target opioid to the brain. In mice and rats, mAb pre-treatment reduced fentanyl-induced respiratory depression and bradycardia, two risk factors for opioid-related overdose fatality. Overall, these results support use of mAb to counteract toxic effects of opioids and other chemical threats. Significance Statement The incidence of fatal overdoses due to the widespread access to heroin, prescription opioids, and fentanyl suggests that current FDA-approved countermeasures are not sufficient to mitigate the opioid epidemic. Monoclonal antibodies (mAb) may provide acute protection from overdose by binding to circulating opioids in serum. Use of mAb prophylactically, or post-exposure in combination with naloxone, may reduce hospitalization and increase survival.
A 55-year old male presented with spontaneous and stimulus-triggered tetanic-like activity of the whole body without losing consciousness or orientation (video, http://links.lww.com/CPJ/A196 ). There was no history of trauma, and clinical examination did not reveal any tetanus-prone wound. Electroencephalography showed no significant alteration in brain activity. Serial intravenous benzodiazepines showed limited effect. Frequency and duration of muscle contractions increased over time and led to severe hypoxemia requiring intubation and sedation with propofol for almost 24 hours followed by continuous infusion of midazolam. Daily sedation pauses revealed ongoing generalized muscle spasm triggered by tactile and auditory stimuli and severe vegetative dysregulation. Tetanus antibody level suggested long-term protection. On day 6, toxicology revealed toxic levels of strychnine in serum (180 ng/ml [toxicity > 75 ng/ml]) and urine (514 ng/ml [no toxicity level defined]). Repeated testing showed markedly declined strychnine levels in serum (2.8 ng/ml) and urine (1.1 ng/ml) on day 11. Sedation was ultimately stopped on day 10. Subsequently, the patient recovered completely and was discharged on day 25. Strychnine ingestion mode remained unclear. Generalized tetanic spasm with sustained alertness, presumably caused by selective inhibition of post-synaptic glycine receptors in the spinal cord, should trigger testing for strychnine poisoning. 1,2 High-dose intravenous benzodiazepines are considered first-line therapy for controlling muscle spasms; however, in patients whose muscle contractions are refractory to benzodiazepines, sedation with propofol or barbiturates is deemed necessary.
Ricin toxin, a plant-derived, mannosylated glycoprotein, elicits an incapacitating and potentially lethal inflammatory response in the airways following inhalation. Uptake of ricin by alveolar macrophages (AM) and other pulmonary cell types occurs via two parallel pathways: one mediated by ricin’s B subunit (RTB), a galactose-specific lectin, and one mediated by the mannose receptor (MR;CD206). Ricin’s A subunit (RTA) is a ribosome-inactivating protein that triggers apoptosis in mammalian cells. It was recently reported that a single monoclonal antibody (MAb), PB10, directed against an immunodominant epitope on RTA and administered intravenously, was able rescue Rhesus macaques from lethal aerosol dose of ricin. In this study, we now demonstrate in mice that the effectiveness PB10 is significantly improved when combined with a second MAb, SylH3, against RTB. Mice treated with PB10 alone survived lethal-dose intranasal ricin challenge, but experienced significant weight loss, moderate pulmonary inflammation (e.g., elevated IL-1 and IL-6 levels, PMN influx), and apoptosis of lung macrophages. In contrast, mice treated with the PB10/SylH3 cocktail were essentially impervious to pulmonary ricin toxin exposure, as evidenced by no weight loss, no change in local IL-1 and IL-6 levels, retention of lung macrophages, and a significant dampening of PMN recruitment into the bronchoalveolar lavage (BAL) fluids. The PB10/SylH3 cocktail only marginally reduced ricin binding to target cells in the BAL, suggesting that the antibody mixture neutralizes ricin by interfering with one or more steps in the RTB- and MR-dependent uptake pathways.
Toxin ingestion is a significant public health issue for children, accounting for hundreds of visits per day to emergency departments. The most common substances are household cleaning products, personal care products, and medications. This article describes an ingestion of a rare substance called strychnine, which is a plant-based odorless powder that was previously used as a nonspecific stimulant. This toxicity can mimic other ingestions, thus delaying diagnosis, so an increased awareness of the common symptoms and laboratory findings may lead to a more targeted management of strychnine poisoning. [Pediatr Ann. 2019;48(5):e205-e207.].
Administration of direct oral anticoagulants (DOACs) for the treatment of venous thrombotic events (VTE) or non-valvular atrial fibrillation (AF) is now standard of care and has demonstrated clinical efficacy and safety in numerous clinical studies. Usually these substances have lower overall mortality and less risk of cerebral hemorrhage, but depending on the substance and study, they are more likely to cause gastrointestinal bleeding than vitamin K antagonists (VKA), the medication that used to be standard for VTE and AF. Since DOACs have very short plasma elimination half-lives compared to VKA, for most bleeding events, expert opinions suggest that withdrawal of DOACs and supportive care will likely suffice to stop a bleeding episode. Because there is a bleeding risk associated with DOACs, reversal strategies may be needed if a patient receiving DOAC therapy bleeds during surgery or an invasive procedure. So far, idarucizumab has been the only available antidote that binds specifically to dabigatran and safely and quickly reverses its anticoagulant effects. Idarucizumab has no effects on anti Xa inhibitors or other anticoagulants. To date, treatment of serious, life-threatening bleeds in patients with anti-Xa-inhibitor has involved 4 factor prothrombin complex concentrates (PCC). PCC restores normal hemostasis laboratory values in most patients with major bleeding events after anti Xa inhibitor intake. Recently, the US Food and Drug Administration (FDA) approved andexanet alfa as the first specific antidote for the anti-Xa inhibitors apixaban and rivaroxaban. So far clinical experience with this substance and data comparing it with PCC are lacking. Currently ciraparantag is under investigation as a universal reversal agent for all DOACs and low molecular weight heparin as well. Because it is so broadly applicable, ciraparantag might be a good future option for the management of most bleeding complications under anticoagulant treatment. The aim of this review is to summarize recent study data and recommendations on nonspecific and specific DOAC reversal strategies and to present the current evidence.
Background:
Injection of botulinum toxin type A for cosmetic purposes is common. It is believed to be safe, but adverse reactions have been reported, including dysphagia, generalized paralysis, respiratory depression, and death caused by focal injection of the toxin. Early administration of antitoxin in patients with adverse reactions is the mainstay of management, but the time window for its clinical efficacy is not well defined.
Case reports:
Two female adult patients with clinical botulism after botulinum toxin type A injection are described. Both patients had received intramuscular injection of botulinum toxin type A in their calves at beauty shops for cosmetic reasons. They developed clinical botulism about 3 days postinjection. They presented late to the emergency department. Monovalent type A botulinum antitoxin was administered 7 and 9 days from symptom onset, respectively. Both patients showed clinical improvement after the antitoxin treatment. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Patients may present to the emergency department with systemic effects of botulinum toxin type A after cosmetic injection. Clinical efficacy of botulinum antitoxin treatment was observed in two patients who were given the drug 7 and 9 days after the occurrence of symptoms of botulism after cosmetic injection of botulinum toxin type A. It may be worthwhile to commence antitoxin treatment even if patients present late.
Theanine, an additive, holds several effects on the central nervous system without toxicity and affects CNS drugs. Theanine bilaterally alters β wave of the EEG with or without caffeine and pentobarbital-induced locomotor activity. Theanine also enhances hypnosis of pentobarbital sodium (PB) and antidepression of midazolam, suggesting there are complicated interactions between theanine and CNS drugs. On the other side, theanine induces glycine release. Glycine potentiates the strychnine toxicity via NMDA receptor activation. Moreover, PB facilitates GABAA receptor activation by GABA, and it is commonly prescribed for strychnine poison. However, what the role that theanine plays in the anticonvulsion of PB against strychnine poison is still unknown.
Theanine, pentobarbital sodium or strychnine was injected intraperitoneally. EEG was monitored by BIOPAC 16 EEG amplifiers. LD50 of strychnine and hypnotic ED50 of pentobarbital sodium with or without theanine for mice were tested according to Bliss' case.
(1) Theanine enhanced the strychnine toxicity. Both theanine and strychnine 1.0 mg/kg increased the power of the β wave. Theanine aggravated that of strychnine 1.0 mg/kg. Theanine attenuated the LD50 of strychnine. (2) Theanine enhanced the anticonvulsion of PB. Theanine increased the power of α, β wave and decreased hypnotic ED50 of PB; PB attenuated strychnine-induced EEG excitation and mortality with or without theanine, and theanine enhanced the effects of PB. Further, theanine enhanced the anticonvulsion of PB dose-dependently against the strychnine toxicity but not the lethal toxicity of strychnine.
These results indicated theanine interacted with PB and strychnine. Theanine enhanced both the strychnine toxicity and anticonvulsion of PB against strychnine poison.
Digoxin poisoning still remains a common cause of morbidity and mortality. Fortunately, digoxin-specific Fab fragments are commercially available as an antidote. However, these Fab fragments are several thousand dollars per vial. There is a standardized formula to calculate appropriate Fab fragment dosage based on the serum digoxin concentration. This can greatly reduce the amount of Fab fragment administered. There is also an empiric dosing guideline recommending 6-10 vials be given; however, this may result in higher amounts of Fab fragments being administered than required. We performed this study to assess the amounts of digoxin-specific Fab fragments administered in the treatment of digoxin poisonings recorded in a poison control system database from January 1, 2000, to December 31, 2009, in which digoxin serum concentrations were available. This was a retrospective study of 278 patients, 107 with acute poisonings (group A) and 171 following chronic poisoning (group B). In group A, the calculated Fab dose was higher than the calculated dose based on available concentrations in 39 (36%) of group A and 15 (9%) of group B patients. The average wholesale price cost of the excessive dosages ranged from 50,589 per patient. Our data suggests that clinician education on digoxin poisoning and the use of the standardized formula to calculate the Fab dose may decrease over utilization and decrease costs associated with the administration of digoxin-specific Fab fragments in the treatment of digoxin poisonings.
Study objectives:
Widow spiders of the genus Latrodectus are found worldwide and produce similar clinical envenomation syndromes. In Australia, red-back spider antivenom (RBS-AV) is effective therapy for Latrodectus hasselti envenomation and it has been reported to reverse envenomation by other widow spiders. This study assessed the efficacy of RBS-AV in preventing in vitro and in vivo toxicity of widow spider venoms of North America and Europe.
Methods:
The binding of RBS-AV to alpha-latrotoxin and Latrodectus venoms (Latrodectus spp mactans, hesperus, lugubris, tredecimguttatus, hasselti) was assayed using Western blotting. Prevention of in vitro toxicity to alpha-latrotoxin and the same venoms was tested by pretreating an isolated chick biventer cervicis nerve-muscle preparation with RBS-AV. Prevention of in vivo toxicity was determined by a lethality study in male Balb/c mice (2.5 to 5x median lethal dose [LD50]) or alpha-latrotoxin (10x LD50) preincubated with antivenom or without RBS-AV (control).
Results:
In Western blots, RBS-AV bound to alpha-latrotoxin and similar widow spider proteins in all venoms tested, indicating antigenic similarity with proteins found in RBS venom. Antivenom prevented the typical in vitro muscle contracture and loss of twitch tension seen with alpha-latrotoxin and the venoms tested. Control mice rapidly developed signs of envenomation, but mice treated with RBS-AV remained free of signs of envenomation.
Conclusion:
RBS-AV prevented both in vitro and in vivo toxicity from Latrodectus venoms and alpha-latrotoxin in mice. These data suggest that RBS-AV may be clinically effective in the treatment of envenomation resulting from the bite of other widow spiders.
Vaccination against nicotine is under investigation as a treatment for tobacco dependence. Passive immunization with nicotine-specific antibodies represents a complementary strategy to vaccination. A potential adverse effect of passive immunization in nicotine-dependent individuals is that it may lead to a rapid reduction in brain nicotine levels and trigger withdrawal. The goal of this study was to determine if passive immunization with the nicotine-specific monoclonal antibody Nic311 precipitated withdrawal in nicotine-dependent rats as measured by increases in brain reward thresholds and somatic signs. Another cohort of rats was used to measure brain nicotine levels after Nic311 administration. Nic311 30, 80 or 240 mg/kg reduced brain nicotine concentrations by 45, 83 or 92% compared to controls. None of these Nic311 doses precipitated withdrawal measured at intervals up to 72 h following antibody administration. Administration of the nicotinic antagonist mecamylamine precipitated a robust nicotine withdrawal syndrome. Therefore, a substantial, but not complete, acute reduction in brain nicotine levels following passive immunization was not sufficient to precipitate nicotine withdrawal in nicotine-dependent rats. The Nic311 doses used have been shown to attenuate the behavioral effects of nicotine, suggesting that the use of passive immunization to treat nicotine addiction is not likely to precipitate withdrawal.
The fresh leaves of Cymbopogon citratus are a good source of an essential oil (EO) rich in citral, and its tea is largely used in the Brazilian folk medicine as a sedative. A similar source of EO is Cymbopogon winterianus, rich in citronellal. The literature presents more studies on the EO of C. citratus and their isolated bioactive components, but only a few are found on the EO of C. winterianus. The objective of the present study was then to study, in a comparative way, the effects of both EOs on three models of convulsions (pentylenetetrazol, pilocarpine, and strychnine) and on the barbiturate-induced sleeping time on male Swiss mice. The animals (20-30 g) were acutely treated with 50, 100, and 200 mg kg(-1), intraperitoneally, of each EO, and 30 min later, the test was initiated. The observed parameters were: latency to the first convulsion and latency to death in seconds. Furthermore, the in vitro effects of the EOs were also studied on myeloperoxidase (MPO; a biomarker for inflammation) and lactate dehydrogenase (LDH; an index of cytotoxicity) releases from human neutrophils. The EOs radical-scavenging activities were also evaluated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The results showed that both EOs were more active on the pentylenetetrazol-induced convulsion model, and C. citratus was even more efficient in increasing latency to the first convulsion and latency to death. Both parameters were potentiated in the presence of a lower dose of diazepam (reference drug) when associated to a lower dose of each EO (25 mg kg(-1)). Besides, their anticonvulsant effects were blocked by flumazenil, a known benzodiazepine antagonist. This effect was somewhat lower on the pilocarpine-induced convulsion, and better effects were seen only with the EOs' higher doses (200 mg kg(-1)). A similar result was observed on the strychnine-induced convulsion model. Both EOs potentiated the barbiturate-induced sleeping time. However, C. citratus was more efficient. Interestingly, both EOs completely blocked the MPO release from human neutrophils and showed no cytotoxic effect on the LDH release from human neutrophils. On the other hand, only a very low or no effect on the DPPH assay was observed with C. winterianus and C. citratus, respectively, indicating that the radical scavenging activity did not play a role on the EOs' effects. We conclude that the mechanism of action of the anticonvulsant effect of the EOs studied is, at least in part, dependent upon the GABAergic neurotransmission. In addition, their effects on inflammatory biomarkers can also contribute to their central nervous system activity.
1. Strychnine-sensitive glycine receptors are primarily localized in the brainstem and spinal cord where they are the major mediators of postsynaptic inhibition. A compound which acts functionally like a glycine receptor agonist would be potentially useful as a pharmacological tool and as a therapeutic agent for treating disorders of glycinergic transmission. 2. MDL 27,531 (4-methyl-3-methylsulphonyl-5-phenyl-4H-1,2,4-triazole) blocked strychnine-induced tonic extensor seizures in mice following either intraperitoneal (ED50 = 12.8 mg kg-1; 30 min) or oral (ED50 = 7.3 mg kg-1; 30 min) administration. Time course studies revealed a maximal effect at 30-60 min, though significant activity was still seen after 24 h. 3. MDL 27,531 was selective in antagonizing strychnine seizures and little or no activity was seen against seizures produced by other chemical convulsants (bicuculline; quinolinic acid; mercaptopropionic acid); by electrical stimuli (maximal electroshock); or by sensory stimuli (audiogenic seizure susceptible mice). MDL 27,531 blocked pentylenetetrazol-induced seizures with an ED50 = 55 mg kg-1. This profile differed from those of the anticonvulsants diazepam, valproic acid, and diphenylhydantoin. 4. The antagonism of strychnine seizures by MDL 27,531 occurred at doses that did not produce signs of sedation (suppression of spontaneous motor activity), motor ataxia (disruption of rotarod performance), muscle relaxation (inhibition of morphine-induced Straub tail), or CNS depression (potentiation of hexobarbitone sleep time). MDL 27,531 had less side effect potential (as derived from ratios obtained from the above measures) relative to those of the known muscle relaxants diazepam and baclofen. 5. Although MDL 27,531 behaved functionally like a selective agonist at the strychnine-sensitive glycine receptor, the compound did not alter the in vitro binding of [3H]-strychnine to mice brainstem/spinal cord membranes at concentrations of up to 100 microM. In further in vitro binding assays, MDL 27,531 at concentrations of up to 100 microM, did not displace the binding of [3H]-muscimol, [3H]-flunitrazepam, or["S]-t-butylbicyclophosphorthionate to rat cortical membranes. These ligands bind to the 7y-aminobutyric acid (GABA), benzodiazepine, and picrotoxin-convulsant binding sites, respectively.6. MDL 27,531 (10-100mgkg-', i.p.) enhanced binding of the benzodiazepine antagonist [3H]-Ro15-1788 to mouse cerebral cortex in vivo without directly affecting GABA levels.7. Ro 15-1788 (16, 32 mg kg-') significantly blocked the MDL 27,531 antagonism of strychnineinduced seizures, though this antagonism was not competitive. The same doses of Ro 15-1788 produced parallel rightward shifts in the dose-response curves for diazepam inhibition of pentylenetetrazol-induced seizures, consistent with a competitive antagonism.8. Thus, MDL 27,531 acts functionally like an agonist at the strychnine-sensitive glycine receptor in its capacity to reverse selectively strychnine-induced seizures. Though the precise mechanism of action of MDL 27,531 is unknown, MDL 27,531 may act at an allosteric site on the strychnine-sensitive receptor which produces agonist-like activity.
Strychnine poisoning is an unusual but dramatic poisoning in which convulsions are the major threat to life. Convulsions are predominantly at the spinal level, and the key to recognition of this poisoning is observation of convulsive activity in the awake patient without a postictal phase. Successful treatment requires aggressive airway control and treatment of seizures with benzodiazepines or barbiturates. Neuromuscular blockade may be required. Gastrointestinal decontamination is usually indicated in recent acute ingestions but may precipitate convulsions. Recovery from strychnine poisoning is usually complete and rapid if treatment is aggressive. In the absence of trauma, compartment syndrome, rhabdomyolysis, or anoxic central nervous system injury, no neurologic or musculoskeletal sequelae are expected. Confirmation of strychnine poisoning is best obtained by submitting urine or gastric aspirate for analysis utilizing a qualitative test such as thin layer chromatography (TLC).
Polyclonal antibodies have been raised in rabbits against the glycine receptor antagonist strychnine, coupled through a 2-amino substituent to the antigenic protein key-hole limpet haemocyanin. Strychnine binding of the predominantly immunoglobulin G (IgG) class of antibodies was measured by incubation with [3H]strychnine, followed by adsorption of IgG onto Staphylococcus aureus cells and filtration through glass-fibre filters under vacuum. Only strychnine and structurally related alkaloids or derivatives were able to inhibit [3H]strychnine binding to the IgG. A significant rank correlation was found between the potencies of these compounds to inhibit [3H]strychnine binding to the antibodies and to the glycine receptor in mouse spinal cord membranes. In contrast, preincubation of strychnine antibodies with a variety of ligands at other neurotransmitter, drug, or hormone receptors in the CNS (at 10(-4) M) failed to inhibit binding significantly. The failure of glycine to inhibit strychnine antibody binding is consistent with previous suggestions that the recognition sites for this amino acid on the CNS receptor may be conformationally distinct from those for the antagonist alkaloid. Strychnine antibodies may now help in the identification and purification of possible endogenous ligands at this alkaloid binding site in the CNS.
The successful supportive management of an acute strychnine ingestion is described; clinical and biochemical findings are reported. A gas chromatographic assay for serum strychnine has been applied to the analysis of 19 serial blood samples. Four hours post ingestion, the serum strychnine concentration was 1.6 mg/L. This declined with T 1/2 [corrected] of 10 hours over a period of 50 hours. First order kinetics are indicated. Management of strychnine poisoning in general is discussed in the light of this case and the limited knowledge of the pharmacokinetics of the alkaloid in man.
1. The effects of some noradrenergic agents on seizures induced by strychnine were investigated in mice. 2. Strychnine (0.5-4 mg/kg, i.p.) dose-dependently produced tonic seizures. 3. DOPS (4-8 mg/kg, i.p.) significantly shortened the latency of seizures elicited by strychnine (2 mg/kg, i.p.). Similarly, DOPS (4 mg/kg, i.p.) effectively increased the incidence and significantly shortened the latency of seizures induced by strychnine (1 mg/kg, i.p.). 4. Imipramine (20-40 mg/kg, i.p.) and pargyline (200 mg/kg, i.p.) significantly shortened the latency of strychnine (2 mg/kg, i.p.)-induced seizures. 5. Phentolamine (5-20 mg/kg, i.p.) effectively antagonised the seizures elicited by strychnine (2 mg/kg, i.p.). Furthermore, phentolamine (10 mg/kg, i.p.) attenuated the seizure-potentiating effect of DOPS (4 mg/kg, i.p.). 6. Propranolol (0.5-2 mg/kg, i.p.) and prazosin (1-2 mg/kg, i.p.) reduced the incidence and significantly delayed the latency of seizures induced by strychnine (2 mg/kg, i.p.). 7. Reserpine (5-10 mg/kg, i.p.) significantly prolonged the latency of strychnine (2 mg/kg, i.p.)-induced seizures. 8. Clonidine (0.25-1 mg/kg, i.p.) dose-dependently and significantly antagonised strychnine (2 mg/kg, i.p.)-induced seizures. 9. Idazoxan (1-4 mg/kg, i.p.) in a dose related manner significantly shortened the latency of seizures induced by strychnine (2 mg/kg, i.p.). Similarly, idazoxan (2 mg/kg, i.p.) profoundly potentiated seizures elicited by strychnine (1 mg/kg, i.p.). Idazoxan (4 mg/kg, i.p.) significantly antagonised the protective effect of clonidine (1 mg/kg, i.p.) against strychnine (2 mg/kg, i.p.)-induced seizures. 10. Disulfiram (3 x 25 - 3 x 100 mg/kg, i.p.) significantly attenuated strychnine (2 mg/kg, i.p.)-induced seizures. DOPS (4 mg/kg, i.p.) significantly potentiated strychnine seizures in disulfiram (3 x 100 mg/kg, i.p.)-pretreated animals. 11. These results indicate that enhancement of noradrenergic neurotransmission potentiates strychnine seizures in mice.
Strychnine competes with the inhibitory neurotransmitter glycine producing an excitatory state characterized clinically by hyperreflexia, severe muscle spasms, and convulsions. However, the kinetics after overdose have not been well described. Case Report: A 34 year-old male presented to the emergency department 20 minus after ingesting half of a 250-mL container of 2% strychnine sulfate (2.25 g). The reported lethal dose is 100-120 mg. He was alert and oriented and experiencing muscle spasms. His condition deteriorated prompting sedation, muscle paralysis, and tracheal intubation. He was given activated charcoal 100 g per nasogastric tube. He was admitted to intensive care where he was managed with diazepam, pentobarbital, and pancuronium. Despite mild rhabdomyolysis, he recovered and was extubated on day three. Although receiving prophylactic heparin therapy, a massive fatal pulmonary embolus ensued. Eighteen blood specimens for strychnine analysis were obtained from 20 minutes to 51 hours after ingestion. Serum concentrations were determined with gas chromatography-mass spectroscopy. Disappearance followed a first-order process with a t 1/2 of 16 hours (r, = 0.97).
Our results confirm the findings of an earlier case report of 19 strychnine levels obtained between 4 and 19 hours which described first-order kinetics with a similar t 1/2 of 10 hours.
Strychnine disappearance in this overdose was well described by a first-order process with a t 1/2 of 10-16 hours.
Case report:
An 18-year-old female who accidentally ingested strychnine developed chemical pancreatitis in addition to the classical clinical picture of strychnine poisoning. Many drugs or chemicals have been reported to be associated with pancreatitis; however, this paper provides us with the first evidence that acute pancreatitis may follow strychnine poisoning. The patient survived despite the development of seizures, lactic acidosis, rhabdomyolysis, and pulmonary infiltrates. Toxicology testing confirmed the presence of strychnine in blood (2.17 mg/L), gastric aspirate, and urine. Attention is drawn to the fact that survival can follow the ingestion of large doses of strychnine providing there is no delay in diagnosis and treatment. The pathophysiologic mechanism of chemical pancreatitis is discussed.
Strychnine poisoning is uncommon, and in most severe cases, the patient dies before reaching hospital. The management of strychnine poisoning is well documented, although there are few data on the kinetics of elimination of strychnine after overdose.
A 42-year-old man presented shortly after ingestion of an unknown quantity of strychnine powder. After a respiratory arrest, with intensive supportive management requiring admission to an intensive care unit, he survived. Eight serum samples were taken over the first 5 days and analysed subsequently for strychnine concentrations.
The initial concentration at 1.5 hours after ingestion was 4.73 mg/l, falling to 0.38 mg/l at 74 hours postingestion. Serum concentrations followed a monoexponential elimination curve with a calculated elimination half-life of 12 hours.
Strychnine poisoning presents with classical features, and with early diagnosis and supportive management, the patient can survive. The initial serum concentration of 4.73 mg/l is the highest reported concentration in a patient who has survived. Previous reports of the elimination half-life have suggested it is between 10 and 16 hours, which conforms to the elimination data in our case.