Antivenom effect on lymphatic absorption and pharmacokinetics of coral snake venom using a large animal model.

Context: Historically, administration and dosing of antivenom (AV) have been guided primarily by physician judgment because of incomplete understanding of the envenomation process. As demonstrated previously, lymphatic absorption plays a major role in the availability and pharmacokinetics (PK) of coral snake venom injected subcutaneously, which suggests that absorption from subcutaneous tissue is the limiting step for venom bioavailability, supporting the notion that the bite site is an ongoing venom depot. This feature may underlie the recurrence phenomena reported in viperid envenomation that appear to result from a mismatch between venom and AV PK. The role of lymphatic absorption in neutralization of venom by AV administered intravenously remains unclear. Methods: The effect of AV on systemic bioavailability and neutralization of Micrurus fulvius venom was assessed using a central lymph-cannulated sheep model. Venom was administered by subcutaneous injection in eight sheep, four with and four without thoracic duct cannulation and drainage. Two hours after venom injection, AV was administered intravenously. Venom and AV concentrations in serum and lymph were determined by ELISA assay from samples collected over a 6-h period and in tissues harvested post-mortem. Results: After AV injection, venom levels in serum fell immediately to undetectable with a subsequent increase in concentration attributable to non-toxic venom proteins. In lymph, AV became detectable 6 min after treatment; venom levels dropped concurrently but remained detectable 4 h later. Post-mortem samples from the venom injection site confirmed the presence of venom near the point of injection. Neither venom nor AV was detected at significant concentrations in major organs or contralateral skin. Conclusions: Intravenous AV immediately neutralizes venom in the bloodstream and can extravasate to neutralize venom absorbed by lymph but this neutralization seems to be slow and incomplete. Residual venom in the inoculation site demonstrates that this site functions as a depot where it is not neutralized by AV, which allows the venom to remain active with slow delivery to the bloodstream for ongoing systemic distribution.

Protein content of antivenoms and relationship with their immunochemical reactivity and neutralization assays.

CONTEXT: Therapy for snakebites relies on the application of antivenoms, which may be produced with different immunogenic mixtures of venom and possess different pharmaceutical characteristics. For these reasons, immunological cross-reactivity and heterologous neutralization were analyzed relative to the protein content of three antivenoms used in the Americas. METHODS: The antivenoms studied were composed of equine F(ab')2 fragments from animals immunized with Crotalinae venoms. The antivenoms were tested against venoms of seven pit viper species from Argentina, seven from Mexico, one from Costa Rica, and one from Colombia. RESULTS: Immunoblotting showed high cross-reactivity of all major protein bands with all the antivenoms tested. ELISA results also showed high cross-reactivity among the different venoms and antivenoms, and a high heterologous neutralization was observed. The results can be interpreted in different ways depending on whether the reactivity is considered in terms of the volume of antivenom used or by the amount of protein contained in this volume of antivenom. The antivenoms with high immunochemical reactivity and neutralizing capacity were those with higher protein content per vial; but when doses were adjusted by protein content, antivenoms of apparently lower neutralizing capacity and immunochemical reactivity showed at least similar potency and reactivity although volumetrically at higher doses. CONCLUSION: Protein content relative to neutralization potency of different products must be taken into account when antivenoms are compared, in addition to the volume required for therapeutic effect. These results show the importance of obtaining high-affinity and high-avidity antibodies to achieve good neutralization using low protein concentration and low-volume antivenoms.

Effectiveness of Centruroides scorpion antivenom compared to historical controls.

BACKGROUND: Envenomation by North American scorpions of genus Centruroides is associated with a syndrome of neurotoxicity and respiratory compromise that disproportionately affects rural children. Severe scorpion envenomation is rare, which makes treatment difficult to study using conventional controlled clinical trials; and small-scale placebo-controlled trials conducted in tertiary centers are of limited generalizability to the community setting. Open label studies, although safer and easier to conduct, are of limited value unless a suitable comparator group is used. Historical controls may be appropriate when concurrent controls are not feasible or ethical. METHODS: A successful placebo-controlled, double-blind clinical trial design was adapted for community use in Arizona and Mexico. A comparator population was established by replacement of the placebo group with a retrospective cohort and preservation of criteria for inclusion, exclusion, dosing and endpoint assessment. Study endpoints were selected to demonstrate the clearest possible difference between treatment groups, while minimizing confounders. Results were summarized and endpoints were directly compared between groups and with the prior double-blind study. RESULTS: The clinical syndrome remained evident in 95.9% of the historical cohort (93/97) 4 h after admission, and their cumulative dose of midazolam given between baseline and discharge was 5.29 ± 8.68 mg/kg (range 0-62.8). Among 78 prospectively treated cases, none received midazolam and only 2 (2.8%) remained symptomatic at 4 h. Venom was detectable in the plasma of all antivenom recipients tested, and it dropped by 90% of baseline in 95% of cases studied. CONCLUSIONS: The results of this pragmatic study strongly support the findings of the double-blind, placebo controlled clinical trial of the same antivenom. Recipients of antivenom at rural sites improved at a rate similar to that in the intensive care (ICU) setting, and historical cases resolved at a rate similar to that for untreated ICU controls. Use of antivenom in the primary care setting appeared to be safe and effective and to eliminate the need for intensive care or for transport to a tertiary care center, in all subjects prospectively studied.

Lymphatic route of transport and pharmacokinetics of Micrurus fulvius (coral snake) venom in sheep.

The contribution of the lymphatic system to the absorption and systemic bioavailability of Micrurus fulvius venom after subcutaneous (SC) administration was assessed using a central lymph-cannulated sheep model. Micrurus fulvius venom was administered either by intravenous bolus (IV) or subcutaneous injection (SC) in 12 sheep with and without thoracic duct cannulation and drainage. Venom concentration in serum and lymph was determined by a sandwich enzyme-linked immunosorbent assay (ELISA) in samples collected over a 6-hour period and in tissues harvested at the end of the experiment. Pharmacokinetic parameters were determined by a non-compartmental analysis. In the lymphatic cannulated group, over the 6 hours after the venom was administered, 69% of administered dose was accounted for in blood (45%) and lymph (25%). Negligible levels of venom were detected in organs and urine implying that the steady state observed after SC administration is maintained by a slow absorption process. Comparison of kinetics of the thoracic duct cannulated and non-cannulated groups showed that lymphatic absorption contributed in an important way to maintenance of this steady state. These results show that the limiting process in the pharmacokinetics of Micrurus fulvius venom following SC administration is absorption, and that the lymphatic system plays a key role in this process.

Pharmacokinetics in rabbits and anti-sphingomyelinase D neutralizing power of Fab, F(ab')(2), IgG and IgG(T) fragments from hyper immune equine plasma.

We describe the separation of whole IgG, IgG(T)-less IgG (called here merely IgG) and IgG(T) and the production of Fab and F(ab')(2) fragments. We studied the pharmacokinetics of these immunoglobulins and fragments in rabbits. Both, the isotypes and the whole IgG fragments were purified and/or produced from the same plasma lot from horses hyper immunized against sphingomyelinase D to produce anti-Loxosceles antivenom. The sphingomyelinase D neutralizing ability of the isotypes and their fragments was measured. Fab and F(ab')(2) PK was well described by a tri-exponential kinetics. IgG and IgG(T) PK, however, deviated from the tri-exponential kinetics 120h after injecting a bolus of the immunotherapeutics. The departure between tri-exponential PK and the experimental data, was shown to be due to a surge of anti-horse antibodies occurring after 120h, peaking at approximately 260h and decaying slowly afterward.

[Envenomation and poisoning by venomous or poisonous animals. VII: arachnidism in the New World]. / Envenimations et empoisonnements par les animaux venimeux ou vénéneux. VII: l'arachnidisme du Nouveau Monde.

The incidence of scorpion stings and spider bites is high in Latin America. This is particularly true for Mexico, part of Amazonia, and southern and eastern Brazil. Centruroides and Tityus scorpion stings present a real danger for humans, especially children. Envenomation results in intense pain, neurological signs, and cardiorespiratory manifestations that can lead to death by acute pulmonary edema or heart failure. In the event of confirmed envenomation, antivenin must be administered as soon as possible in association with symptomatic treatment and, if necessary, cardiorespiratory resuscitation. Spider bites are a less frequent and severe. Envenomation by Loxosceles is extremely painful and necrotizing. Severe visceral complications can develop. An effective antivenim has recently become available for local and systemic envenomation. Envenomation by Latrodectus leads to neurological symptoms that can also be treated with antivenom. Envenomation by other spiders is less frequent and generally harmless.

Clinical trial of an F(ab')2 polyvalent equine antivenom for African snake bites in Benin.

We report the results of a trial designed to measure the safety and efficacy of African Antivipmyn, a new freeze-dried polyvalent equine F(ab')(2)-based antivenom. We tested 289 envenomations. After treatment, 19% of treated patients had undesirable events, all benign. A possible adverse effect was attributed to this antivenom in 11% of the patients. Bleeding was observed in 48% of the patients; it stopped within 2 hours after treatment with antivenom in 60% of the patients. Blood incoagulability was observed in 80% of the patients. Restoration of coagulation was attained within 4 hours in 60% of the patients. Nine patients died; 6 arrived at the hospital in the final stage of complications and 5 arrived at the hospital more than 60 hours after the bite. The value of blood coagulation tests in diagnosis of envenomation and bleeding as an indicator of renewal of treatment are emphasized.

Antigenic cross-reactivity between sixteen venoms from scorpions belonging to six genera.

Venoms of 15 scorpion species from Venezuela and one from Brazil were compared in their antigenic cross-reactivity with specific F(ab')2 against Tityus discrepans (Td-antibodies), using the method of King and collaborators (1). Our results show that Tityus venoms cross-reactivity (shared epitopes) with the venoms of other species within the genus tended to be less for a greater distance between the habitat of the species. A nonparametric linear regression of free Td-antibody binding to T. discrepans venom immobilized to a solid phase in the presence of other Tityus venoms versus distance showed binding = a + b x log10 (distance) where: median (95% confidence interval) for a = 0.92 (7.43, 9.80) and b = 17.20 (4.15, 22.57) binding/log10(Km); Spearman rS = 0.783 with associated P = 0.006. Our results show that toxins from different Tityus species, targeting mammalian Na+ and K+ channels, are antigenically very similar. Venoms from species from other genera such as Centruroides, Broteas, Diplocentrus, Chactas, and Rhopalurus did not cross-react with Td-antibodies.

Pharmacokinetics of a F(ab')2 scorpion antivenom in healthy human volunteers.

This paper presents the first study of F(ab')2 scorpion antivenom pharmacokinetics in humans. We have studied the pharmacokinetics of an antiscorpion venom preparation (Alacramyn) in eight human healthy volunteers. The fabotherapic was administered as a 47.5 mg i.v. bolus. Blood samples were drawn at 0, 5, 15, 30, 45, 60, 90, 120, 180 and 360 min after antivenom administration. Subsequently, the volunteers made seven visits to the hospital. Four of them at 24 h intervals, one at day 10, and one at day 21. We measured antivenom plasmatic concentrations using a specific high sensitivity ELISA method for F(ab')2. The time course of F(ab')2 in serum of seven subjects was well described by a lineal combination of three exponential components; a four exponential component model was necessary to fit the eighth subject. The most significant antivenom pharmacokinetic parameters determined were: AUC(infinity)=596.9 (369.3, 891.2) mg h l(-1); V(c) = 3.1 (2.3, 4.3)l; V(ss) = 15.4 (12.8, 39.9)l; MRT = 250.0 (218.8, 310.2) h; CL = 96.6 (58.0, 139.2) ml h(-1); t(1/2,tau1) (also called t(1/2,alpha)) = 0.25 (0.13, 0.37) h; t(1/2,tau(z)) (corresponding to the slowest component) = 161.3 (141.0, 212.0) h.

Relationship between plasmatic levels of various cytokines, tumour necrosis factor, enzymes, glucose and venom concentration following Tityus scorpion sting.

A sandwich enzyme-linked immunosorbent assay was developed for measuring Tityus venom levels in plasma. The method proved capable of distinguishing patients with only local symptoms from controls, and was used to quantify venom in 205 accidental human envenomations. Our results show that the severity of envenoming is related to the patient plasma venom concentration. This depends on time elapsed between the sting and when the plasma was drawn. We observed that 46 and 49% of patients with moderate to severe symptoms (MS, n=41) showed hyperamylasemia and hyperglycemia, respectively. In addition, 39% of cases with MS symptoms had partial thromboplastin time values prolonged or shorted and 6.5% of patients with local symptoms (LS, n=164) had only prolonged prothrombin time values. Interleukin 6 (IL6) increased significantly in patients with MS symptoms. IL6 values increased with hyperamylasemia, envenoming severity and time hyperamylasemia.

Los pacientes de escorpionismo con sintomatología local tiene niveles importantes de veneno en plasma / The scorpionism patients with local symptomatology have important venoms levels in plasma

Se desarrolló un método de inmunoensayo (Enzime-Linked Imunosorbent Assay) sensible y específico para cuantificar veneno de T. discrepans en plasma de pacientes de escorpionismo. Los casos se clasificaron como: asintomáticos (Clasificación del Hospital "Dr. Leopoldo Manrique Terrero", Caracas, D.F.) equivalente a sintomatología local (Clasificación del Hospital "Victorino Santaella", Los Teques, Edo. Miranda), o con sintomatología sistemática: leve, moderada o grave. Se analizó el plasma de 82 pacientes, de los cuales 23 fueron niños. La venenemia en 58 pacientes con sintomatología local estuvo entre 0,01 y 17,2 ng/ml con una mediana e intervalos de confianza de 95 por ciento (entre paréntesis) de 0,5 (0,2 - 2)ng/ml. La venenemia en 13 pacientes con sintomatología leve o moderada flactuó desde 0,1 hasta 202 ng/ml [11,2 (0,5 - 80,4) ng/ml]. Sólo se obtuvo plasma de un paciente con sintomatología grave el cual presentó una venenemia de 31,8 ng/ml. El nivel de falsos positivos del ensayo se determinó con el plasma de 10 personas no emponzoñadas en las cuales el ensayo indicó una venenemia aparente de 0,13 (0,5 - 0,25) ng/ml. Las concentraciones más altas y después de aplicar antiveneno (n= 7) demostró que éste hace desaparecer el veneno del plasma. Se observaron alteraciones en el tiempo parcial de tromboplastina prevalentemente en pacientes con sintomatología local. Alteraciones en amilasa y glicemia sólo se observaron en pacientes con clínica sistémica. El 14 por ciento de 58 pacientes con sintomatología local presentaron alteraciones del tiempo parcial de tromboplastina indicativas de anticoagulación en algunos, o procoagulación en otros casos; estos coincide con resultados in vitro de nuestro laboratorio donde se identificaron fracciones pro y anticoagulantes en el veneno de Tityus discrepans (D'Suze y col., 2000). En 63 por ciento de los pacientes con sintomatología local se detectó veneno en el plasma, 24 por ciento de ellos en concentraciones entre 1 y 4 ng/ml y 9 por ciento por encima de ese rango, estas concentraciones de veneno se correlacionan en otros estudios con aumentos de citokinas, factores de necrosis tumoral y de agregación plaquetaria. Este trabajo indica que la ausencia de clínica no es equivalente a ausencia de veneno en plasma ni a ausencia de daño. Este trabajo fue financiado por el proyecto aplicado IVIC-Silanes

Inhibition of gene expression in Entamoeba histolytica with antisense peptide nucleic acid oligomers.

Peptide nucleic acids (PNAs) may be a potent tool for gene function studies in medically important parasitic organisms, especially those that have not before been accessible to molecular genetic knockout approaches. One such organism is Entamoeba histolytica, the causative agent of amebiasis, which infects about 500 million people and is the cause of clinical disease in over 40 million each year, mainly in the tropical and subtropical world. We used PNA antisense oligomers to inhibit expression of an episomally expressed gene (neomycin phosphorotransferase, NPT) and a chromosomal gene (EhErd2, a homolog of Erd2, a marker of the Golgi system in eukaryotic cells) in axenically cultured trophozoites of E. histolytica. Measurement of NPT enzyme activity and EhErd2 protein levels, as well as measurement of cellular proliferation, revealed specific decreases in expression of the target genes, and concomitant inhibition of cell growth, in trophozoites treated with micromolar concentrations of unmodified antisense PNA oligomers.