An alternative method for comparing hemorrhagic activity of snake venoms following one of the 3R's principles.

Venom-induced hemorrhage analysis usually is performed by Minimum Hemorrhagic Dose (MHD), however a similar method can be used to compare venoms with fewer laboratory animals. Our work compared the MHD of five different venoms, with the size of hemorrhagic spot, finding good correlations in the results. Considering the 3Rs principle, we propose the use of the hemorrhagic spot method to compare hemorrhagic activity of snake venoms, rather than using the MHD method, since the first one needs 5 times less animals than the other.

The Cloning and Characterization of a Three-Finger Toxin Homolog (NXH8) from the Coralsnake Micrurus corallinus That Interacts with Skeletal Muscle Nicotinic Acetylcholine Receptors.

Coralsnakes (Micrurus spp.) are the only elapids found throughout the Americas. They are recognized for their highly neurotoxic venom, which is comprised of a wide variety of toxins, including the stable, low-mass toxins known as three-finger toxins (3FTx). Due to difficulties in venom extraction and availability, research on coralsnake venoms is still very limited when compared to that of other Elapidae snakes like cobras, kraits, and mambas. In this study, two previously described 3FTx from the venom of M. corallinus, NXH1 (3SOC1_MICCO), and NXH8 (3NO48_MICCO) were characterized. Using in silico, in vitro, and ex vivo experiments, the biological activities of these toxins were predicted and evaluated. The results showed that only NXH8 was capable of binding to skeletal muscle cells and modulating the activity of nAChRs in nerve-diaphragm preparations. These effects were antagonized by anti-rNXH8 or antielapidic sera. Sequence analysis revealed that the NXH1 toxin possesses eight cysteine residues and four disulfide bonds, while the NXH8 toxin has a primary structure similar to that of non-conventional 3FTx, with an additional disulfide bond on the first loop. These findings add more information related to the structural diversity present within the 3FTx class, while expanding our understanding of the mechanisms of the toxicity of this coralsnake venom and opening new perspectives for developing more effective therapeutic interventions.

Standard Quality Characteristics and Efficacy of a New Third-Generation Antivenom Developed in Colombia Covering Micrurus spp. Venoms.

In Colombia, Micrurus snakebites are classified as severe according to the national clinical care guidelines and must be treated with specific antivenoms. Unfortunately, these types of antivenoms are scarce in certain areas of the country and are currently reported as an unavailable vital medicine. To address this issue, La Universidad de Antioquia, through its spin-off Tech Life Saving, is leading a project to develop third-generation polyvalent freeze-dried antivenom. The goal is to ensure access to this therapy, especially in rural and dispersed areas. This project aims to evaluate the physicochemical and preclinical parameters (standard quality characteristics) of a lab-scale anti-elapid antivenom batch. The antivenom is challenged against the venoms of several Micrurus species, including M. mipartitus, M. dumerilii, M. ancoralis, M. dissoleucus, M. lemniscatus, M. medemi, M. spixii, M. surinamensis, and M. isozonus, following the standard quality characteristics set by the World Health Organization (WHO). The antivenom demonstrates an appearance consistent with standards, 100% solubility within 4 min and 25 s, an extractable volume of 10.39 mL, a pH of 6.04, an albumin concentration of 0.377 mg/mL (equivalent to 1.22% of total protein), and a protein concentration of 30.97 mg/mL. Importantly, it maintains full integrity of its F(ab')2 fragments and exhibits purity over 98.5%. Furthermore, in mice toxicity evaluations, doses up to 15 mg/mouse show no toxic effects. The antivenom also demonstrates a significant recognition pattern against Micrurus venoms rich in phospholipase A2 (PLA2) content, as observed in M. dumerilii, M. dissoleucus, and M. isozonus. The effective dose 50 (ED50) indicates that a single vial (10 mL) can neutralize 2.33 mg of M. mipartitus venom and 3.99 mg of M. dumerilii venom. This new anti-elapid third-generation polyvalent and freeze-dried antivenom meets the physicochemical parameters set by the WHO and the regulators in Colombia. It demonstrates significant efficacy in neutralizing the venom of the most epidemiologically important Micrurus species in Colombia. Additionally, it recognizes seven other species of Micrurus venom with a higher affinity for venoms exhibiting PLA2 toxins. Fulfilling these parameters represents the first step toward proposing a new pharmacological alternative for treating snakebites in Colombia, particularly in dispersed rural areas, given that this antivenom is formulated as a freeze-dried product.

In-depth immunorecognition and neutralization analyses of Micrurus mipartitus and M. dumerilii venoms and toxins by a commercial antivenom.

In Colombia, the Micrurus genus comprises 30 species, including M. mipartitus and M. dumerilii, which are of major clinical relevance due to their wide geographical distribution and the number of snakebites inflicted by them. These neurotoxic envenomations are characterized by neuromuscular paralysis attributed to venom components such as three-finger toxins (3FTx) and phospholipases (PLA2). Additionally, there is limited information available on the neutralizing coverage of commercially available antivenoms, underscoring the need to perform studies to assess the cross-neutralizing ability of these life-saving products. Therefore, we present an in-depth immunorecognition analysis by the anticoral-INS antivenom from Colombia on the M. mipartitus and M. dumerilii venoms. The antivenom cross-recognized the whole venoms and their components with different intensities. For instance, the antivenom showed better recognition on PLA2s than on 3FTxs in both venoms. Moreover, at doses tested, the antivenom totally neutralized the lethal effect of M. dumerilii venom; however, it did not neutralize this effect induced by M. mipartitus venom and its main toxic components from the southwestern region of the department of Antioquia. Furthermore, the anticoral-INS antivenom displayed better cross-immunorecognition of PLA2-predominant Micrurus venoms than of 3FTx-predominant Micrurus venoms. This highlights the need to include venoms from both types of venom patterns in the immunization mixture to produce antivenoms against coral snakes. Finally, our results suggest the need for further research to optimize the composition of immunizing mixtures for antivenom production and improve their efficacy against coral snake envenomation in Colombia and the Americas.

Knowledge about Snake Venoms and Toxins from Colombia: A Systematic Review.

Colombia encompasses three mountain ranges that divide the country into five natural regions: Andes, Pacific, Caribbean, Amazon, and Orinoquia. These regions offer an impressive range of climates, altitudes, and landscapes, which lead to a high snake biodiversity. Of the almost 300 snake species reported in Colombia, nearly 50 are categorized as venomous. This high diversity of species contrasts with the small number of studies to characterize their venom compositions and natural history in the different ecoregions. This work reviews the available information about the venom composition, isolated toxins, and potential applications of snake species found in Colombia. Data compilation was conducted according to the PRISMA guidelines, and the systematic literature search was carried out in Pubmed/MEDLINE. Venom proteomes from nine Viperidae and three Elapidae species have been described using quantitative analytical strategies. In addition, venoms of three Colubridae species have been studied. Bioactivities reported for some of the venoms or isolated components-such as antibacterial, cytotoxicity on tumoral cell lines, and antiplasmodial properties-may be of interest to develop potential applications. Overall, this review indicates that, despite recent progress in the characterization of venoms from several Colombian snakes, it is necessary to perform further studies on the many species whose venoms remain essentially unexplored, especially those of the poorly known genus Micrurus.

Cardiac Effects of Micrurus corallinus and Micrurus dumerilii carinicauda (Elapidae) Venoms and Neutralization by Brazilian Coralsnake Antivenom and Varespladib.

In this work, we examined the action of two South American coralsnake (Micrurus corallinus and Micrurus dumerilii carinicauda) venoms on rat heart function in the absence and presence of treatment with Brazilian coralsnake antivenom (CAV) and varespladib (VPL), a potent phospholipase A2 inhibitor. Anesthetized male Wistar rats were injected with saline (control) or a single dose of venom (1.5 mg/kg, i.m.) and monitored for alterations in echocardiographic parameters, serum CK-MB levels and cardiac histomorphology, the latter using a combination of fractal dimension and histopathological methods. Neither of the venoms caused cardiac functional alterations 2 h after venom injection; however, M. corallinus venom caused tachycardia 2 h after venom injection, with CAV (given i.p. at an antivenom:venom ratio of 1:1.5, v/w), VPL (0.5 mg/kg, i.p.) and CAV + VPL preventing this increase. Both venoms increased the cardiac lesional score and serum CK-MB levels compared to saline-treated rats, but only the combination of CAV + VPL prevented these alterations, although VPL alone was able to attenuate the increase in CK-MB caused by M. corallinus venom. Micrurus corallinus venom increased the heart fractal dimension measurement, but none of the treatments prevented this alteration. In conclusion, M. corallinus and M. d. carinicauda venoms caused no major cardiac functional alterations at the dose tested, although M. corallinus venom caused transient tachycardia. Both venoms caused some cardiac morphological damage, as indicated by histomorphological analyses and the increase in circulating CK-MB levels. These alterations were consistently attenuated by a combination of CAV and VPL.

Antibodies against a single fraction of Micrurus dumerilii venom neutralize the lethal effect of whole venom.

The coralsnake Micrurus dumerilii (Elapidae) is reported to cause envenomings of medical importance. Previous studies characterized the protein composition of its venom, with phospholipase A2 (PLA2) proteins the most abundant. However, it is unknown which venom components are responsible for its lethal toxicity. Fractionation of M. dumerilii venom from Colombia was carried out using RP-HPLC and each fraction was screened for lethal effect in mice at a dose of 20 µg by intraperitoneal route. Results showed that only one fraction, F9, was lethal. This fraction displayed PLA2 activity, induced indirect hemolysis in vitro, as well as edema and myotoxicity in vivo. SDS-PAGE of unreduced F9 evidenced two bands of 8 and 15 kDa, respectively, consistent with the detection of proteins with masses of 13,217.77 Da, 7144.06 Da, and 7665.55 Da. Tryptic digestion of F9 followed by nESI-MS/MS revealed peptide sequences matching proteins of the three-finger toxin (3FTx) and PLA2 families. Immunization of a rabbit with F9 proteins elicited antibody titers up to 1:10,000 by ELISA. After serum fractionation with caprylic acid, the obtained IgG was able to neutralize the lethal effect of the complete venom of M. dumerilii using a challenge of 2 ×LD50 at the IgG/venom ratio of 50:1 (w/w). In conclusion, present results show that the lethal effect of M. dumerilii venom in mice is mainly driven by one fraction which contains 3FTx and PLA2 proteins. The antibodies produced against this fraction cross-recognized other PLA2s and neutralized the lethal effect of whole M. dumerilii venom, pointing out to the potential usefulness of F9 as a relevant antigen for improving current coral snake antivenoms.

Anti-Neurotoxins from Micrurus mipartitus in the Development of Coral Snake Antivenoms.

In Colombia, the genus Micrurus includes 30 species, of which M. mipartitus and M. dumerilii are the most widely distributed. Micrurus causes less than 3% of the approximately 5000 cases of snakebite per year. The elapid envenomation caused by the snakes from the Micrurus genus, are characterized by the severity of their clinical manifestations, due to the venom neurotoxic components such as three-finger toxins (3FTx) and phospholipases (PLA2). The treatment for snakebites is the administration of specific antivenoms, however, some of them have limitations in their neutralizing ability. A strategy proposed to improve antivenoms is to produce antibodies against the main components of the venom. The aim of this work was to produce an antivenom, using an immunization protocol including the main 3FTx and PLA2 responsible for M. mipartitus lethality. The antibody titers were determined by ELISA in rabbits' serum. The immunized animals elicited a response against toxins and whole venom. The Immunoglobulin G (IgGs) obtained were able to neutralize the lethal effect of their homologous toxins. A combination of antivenom from M. mipartitus with antitoxins improved their neutralizing ability. In the same way, a mixture of anti 3FTx and PLA2 protected the mice from a 1.5 median lethal dose (LD50) of M. mipartitus venom. The results showed that this might be a way to improve antibody titers specificity against the relevant toxins in M. mipartitus venom and indicated that there is a possibility to develop and use recombinant 3FTx and PLA2 toxins as immunogens to produce antivenoms. Additionally, this represents an alternative to reduce the amount of venom used in anti-coral antivenom production.

Partial efficacy of a Brazilian coralsnake antivenom and varespladib in neutralizing distinct toxic effects induced by sublethal Micrurus dumerilii carinicauda envenoming in rats.

In this work, we reported the efficacy of a combination of Brazilian therapeutic coralsnake antivenom (CAV) and varespladib (phospholipase A2 inhibitor - VPL) in partially neutralizing selected toxic effects of Micrurus dumerilii carinicauda coralsnake venom in rats. Venom caused local myonecrosis and systemic neurotoxicity, nephrotoxicity, and hepatotoxicity within 2 h of injection. CAV and VPL administered separately failed to prevent most of these alterations. However, a combination of CAV plus VPL offered variable protection against venom-induced coagulation disturbances, leukocytosis, and renal-hepatic morphological alterations.

Effects of nitric oxide on C2C12 cell inflammatory responses and notexin-induced muscle injury / Efectos del óxido nítrico en las respuestas inflamatorias de las células C2C12 y la lesión muscular inducida por notexina

SUMMARY: Skeletal muscle injury is an acute inflammatory condition caused by an inflammatory response. To reduce inflammatory cell infiltration and relieve skeletal muscle injury, efficient treatment is urgently needed. Nitric oxide is a free radical molecule reported to have anti-inflammatory effects. In this study, we showed that NO could inhibit the inflammatory response of C2C12 cells in vitro and protect rat skeletal muscle injury from notexin in vivo. NO synthase inhibitor (L-NG-Nitroarginine Methyl Este?L-NAME) and NO donor (sodium nitroprusside dehydrate ?SNP) were used to explore the vital role of lipopolysaccharides (LPSs) in LPS-stimulated C2C12 myoblasts.The expression of IL-18 and IL-1b was upregulated by L-NAME and downregulated by SNP, as indicated by the ELISA results. NO can reduce ASC, Caspase-1, and NLRP3 mRNA and protein levels. Furthermore, NO was detected in the rat model. The results of immunohistochemical staining showed that the production of DMD decreased. We conducted qRT-PCR and western blotting to detect the expression of Jo-1, Mi-2, TLR2, and TLR4 on day 6 post injury following treatment with L-NAME and SNP. The expression of Jo-1, Mi-2, TLR2, and TLR4 was upregulated by L-NAME and significantly reversed by SNP. NO can alleviate C2C12 cell inflammatory responses and protect rat skeletal muscle injury from notexin.

RESUMEN: La lesión del músculo esquelético es una afección inflamatoria aguda causada por una respuesta inflamatoria. Para reducir la infiltración de células inflamatorias y aliviar la lesión del músculo esquelético es necesario un tratamiento eficaz. El óxido nítrico es una molécula de radicales libres que tiene efectos antiinflamatorios. En este estudio, demostramos que el ON podría inhibir la respuesta inflamatoria de las células C2C12 in vitro y proteger la lesión del músculo esquelético de rata de la notexina in vivo. El inhibidor de ON sintasa (L-NG-nitroarginina metil este, L-NAME) y el donante de ON (nitroprusiato de sodio deshidratado, SNP) se utilizaron para explorar el papel vital de los lipopolisacáridos (LPS) en los mioblastos C2C12 estimulados por LPS. La expresión de IL- 18 e IL-1b fue regulada positivamente por L-NAME y regulada negativamente por SNP, como indican los resultados de ELISA. El ON puede reducir los niveles de proteína y ARNm de ASC, Caspasa-1 y NLRP3. Además, se detectó ON en el modelo de rata. Los resultados de la tinción inmunohistoquímica mostraron que disminuyó la producción de DMD. Realizamos qRT-PCR y transferencia Western para detectar la expresión de Jo-1, Mi-2, TLR2 y TLR4 el día 6 después de la lesión después del tratamiento con L-NAME y SNP. La expresión de Jo-1, Mi-2, TLR2 y TLR4 fue regulada positivamente por L- NAME y significativamente revertida por SNP. El ON puede aliviar las respuestas inflamatorias de las células C2C12 en ratas, y proteger la lesión del músculo esquelético de la notexina.

In Vivo Cardiotoxic Potential of Micrurus frontalis Venom.

In the present study, we investigated the cardiotoxic potential of Micrurus frontalis venom. Twelve guinea pigs (Cavia porcellus) were distributed in two groups (n = 6), named control and envenomed. Control groups received 0.2 ml of PBS/BSA, while envenomed group received 0.2 ml of the same solution containing 450 µg/kg of M. frontalis venom. Both were intramuscular injections. Electrocardiography, echocardiogram, blood count, and serum biochemistry were performed before and 2 h after inoculation. Necropsy was performed, and histological and ultrastructural analysis of the heart were conducted. First clinical signs were presented as early as 18 min after venom inoculation. All poisoned animals presented flaccid paralysis of both hind and forelimbs, followed by fasciculations and respiratory arrythmia. However, the animals did not die in the first 2 h of poisoning. ECG of the poisoned animals revealed severe ventricular arrythmias, corroborated by reduction of both ejection and shortening fractions, increase in CK, CK-MB, troponin, cardiomyocyte degeneration, fragmentation and mitochondrial damage. M. frontalis venom causes severe heart damage, eliciting both morphological and arrhythmogenic effects after only 2 h of envenomation.

Monoclonal-Based Antivenomics Reveals Conserved Neutralizing Epitopes in Type I PLA2 Molecules from Coral Snakes.

For over a century, polyclonal antibodies have been used to treat snakebite envenoming and are still considered by the WHO as the only scientifically validated treatment for snakebites. Nevertheless, moderate innovations have been introduced to this immunotherapy. New strategies and approaches to understanding how antibodies recognize and neutralize snake toxins represent a challenge for next-generation antivenoms. The neurotoxic activity of Micrurus venom is mainly due to two distinct protein families, three-finger toxins (3FTx) and phospholipases A2 (PLA2). Structural conservation among protein family members may represent an opportunity to generate neutralizing monoclonal antibodies (mAbs) against family-conserved epitopes. In this work, we sought to produce a set of monoclonal antibodies against the most toxic components of M. altirostris venom. To this end, the crude venom was fractionated, and its major toxic proteins were identified and used to generate a panel of five mAbs. The specificity of these mAbs was characterized by ELISA and antivenomics approaches. Two of the generated mAbs recognized PLA2 epitopes. They inhibited PLA2 catalytic activity and showed paraspecific neutralization against the myotoxicity from the lethal effect of Micrurus and Naja venoms' PLA2s. Epitope conservation among venom PLA2 molecules suggests the possibility of generating pan-PLA2 neutralizing antibodies.

In vivo treatment with varespladib, a phospholipase A2 inhibitor, prevents the peripheral neurotoxicity and systemic disorders induced by Micrurus corallinus (coral snake) venom in rats.

In this study, we investigated the action of varespladib (VPL) alone or in combination with a coral snake antivenom (CAV) on the local and systemic effects induced by Micrurus corallinus venom in rats. Adult male Wistar rats were exposed to venom (1.5 mg/kg - i.m.) and immediately treated with CAV (antivenom:venom ratio 1:1.5 'v/w' - i.p.), VPL (0.5 mg/kg - i.p.), or both of these treatments. The animals were monitored for 120 min and then anesthetized to collect blood samples used for haematological and serum biochemical analysis; after euthanasia, skeletal muscle, renal and hepatic tissue samples were collected for histopathological analysis. M. corallinus venom caused local oedema without subcutaneous haemorrhage or apparent necrosis formation, although there was accentuated muscle morphological damage; none of the treatments prevented oedema formation but the combination of CAV and VPL reduced venom-induced myonecrosis. Venom caused neuromuscular paralysis and respiratory impairment in approximately 60 min following envenomation; CAV alone did not prevent the neurotoxic action, whereas VPL alone prevented neurotoxic symptoms developing as did the combination of CAV and VPL. Venom induced significant increase of serum CK and AST release, mostly due to local and systemic myotoxicity, which was partially prevented by the combination of CAV and VPL. The release of hepatotoxic serum biomarkers (LDH and ALP) induced by M. corallinus venom was not prevented by CAV and VPL when individually administered; their combination effectively prevented ALP release. The venom-induced nephrotoxicity (increase in serum creatinine concentration) was prevented by all the treatments. VPL alone or in combination with CAV significantly prevented the venom-induced lymphocytosis. In conclusion, VPL shows to be effective at preventing the neurotoxic, nephrotoxic, and inflammatory activities of M. corallinus venom. In addition, VPL acts synergistically with antivenom to prevent a number of systemic effects caused by M. corallinus venom.

S- and P-type cobra venom cardiotoxins differ in their action on isolated rat heart

Background: The cardiovascular system is one of the first systems to be affected by snake toxins; but not many toxins exert a direct effect on the heart. Cobra venom cardiotoxins are among those few toxins that attack the heart. Although the two cardiotoxin types (S and P) differ in their central-loop structure, it is not known whether they differ in their effect on the mammalian heart. We compared the effects of S- and P-type cardiotoxins, CTÐ¥-1 and CTÐ¥-2, respectively, from the cobra Naja oxiana, on the isolated rat heart. Methods: An isolated rat heart perfused according to the Langendorff technique was used in this study to investigate the activity of cardiotoxins CTX-1 and CTX-2. The following parameters were registered: the left ventricular developed pressure, calculated as the difference between systolic and diastolic pressure in the left ventricle, the end-diastolic pressure, the heart rate, time to maximal end-diastolic pressure (heart contracture), and time to depression of the heart contraction. Results: Both cardiotoxins at the concentration of 5 µg/mL initially produce a slight increase in systolic intraventricular pressure, followed by its rapid decrease with a simultaneous increase in diastolic intraventricular pressure until reaching contracture. CTX-2 blocks cardiac contractions faster than CTX-1; in its presence the maximum diastolic pressure is reached faster and the magnitude of the developed contracture is higher. Conclusion: The P-type cardiotoxin CTX-2 more strongly impairs rat heart functional activity than the S-type cardiotoxin CTX-1, as expressed in its faster blockage of cardiac contractions as well as in more rapid development and greater magnitude of contracture in its presence.(AU)

Cardiotoxic Effects of Micrurus surinamensis (Cuvier, 1817) Snake Venom.

Micrurus surinamensis is a coral snake from the Elapidae family of wide distribution in Amazonia Forest. Its venom contains neurotoxins that induce muscular and respiratory paralysis; however, its cardiovascular action is not yet characterized. The aim of this study was to investigate the cardiotoxic effects caused by M. surinamensis poisoning in rodents. Twelve guinea pigs (Cavia porcellus) were distributed in two groups (n = 6) named as control and envenomed. The control group received 0.2 ml of PBS/BSA via intramuscular injection (IM), while envenomed animals received 0.75 µg of venom per g of body weight, also via IM. Electrocardiographic examination (ECG) and biochemical serum tests were conducted before and 2 h after inoculation. ECG of the envenomed animals revealed severe progressive arrhythmias including atrioventricular block, supraventricular, and ventricular extrasystoles. Serum biochemistry showed significant increase in CK, CK-MB, and LDH enzymes corroborating the skeletal and cardiac muscle damage. Myonecrosis and degeneration were observed in both skeletal and heart muscle; nevertheless, transmission electron microscopy revealed cardiac muscle fibers fragmentation. In conclusion, M. surinamensis venom has a potent cardiotoxic activity eliciting arrhythmogenic effects and heart damage after only 2 h of envenomation.

Venoms of New World Vinesnakes (Oxybelis aeneus and O. fulgidus).

Species of Oxybelis are extremely elongate arboreal snakes that are broadly distributed in the Americas, from extreme southeastern Arizona (USA) to central South America. Primarily feeding on lizards and birds, Oxybelis venoms are poorly known in general, but a prominent taxon-specific three-finger toxin (fulgimotoxin) was isolated from and is a prominent component of O. fulgidus venom; a homolog is also present in O. aeneus venom. As part of ongoing characterization of venoms from rear-fanged snakes, we describe here the composition of two broadly distributed species, O. aeneus and O. fulgidus. Venom proteomes were of very low complexity, and four protein families (LAAO, PIII SVMP, CRiSP and 3FTx) account for more than 90% of total protein composition. Venoms from both species are moderately toxic to mice and to Hemidactylus geckos, but they are nearly an order of magnitude more toxic to Anolis lizards (a native prey species). These results reflect a trend in colubrid venom composition that is becoming increasingly more common: the presence of taxon-specific toxins, specifically three-finger toxins, preferentially targeting lizards and/or birds.

Kinetic and toxicological effects of synthesized palladium(II) complex on snake venom (Bungarus sindanus) acetylcholinesterase

The venom of the krait (Bungarus sindanus), an Elapidae snake, is highly toxic to humans and contains a great amount of acetylcholinesterase (AChE). The enzyme AChE provokes the hydrolysis of substrate acetylcholine (ACh) in the nervous system and terminates nerve impulse. Different inhibitors inactivate AChE and lead to ACh accumulation and disrupted neurotransmission. Methods: The present study was designed to evaluate the effect of palladium(II) complex as antivenom against krait venom AChE using kinetics methods. Results: Statistical analysis showed that krait venom AChE inhibition decreases with the increase of Pd(II) complex (0.025-0.05 µM) and exerted 61% inhibition against the AChE at a fixed concentration (0.5 mM) of ACh. Kinetic analysis using the Lineweaver Burk plot showed that Pd(II) caused a competitive inhibition. The compound Pd(II) complex binds at the active site of the enzyme. It was observed that K m (Michaelis-Menten constant of AChE-ACh into AChE and product) increased from 0.108 to 0.310 mM (45.74 to 318.35%) and V max remained constant with an increase of Pd(II) complex concentrations. In AChE K Iapp was found to increase from 0.0912 to 0.025 µM (29.82-72.58%) and did not affect the V maxapp with an increase of ACh from (0.05-1 mM). K i (inhibitory constant) was estimated to be 0.029µM for snake venom; while the K m was estimated to be 0.4 mM. The calculated IC50 for Pd(II) complex was found to be 0.043 µM at constant ACh concentration (0.5 mM). Conclusions: The results show that the Pd(II) complex can be deliberated as an inhibitor of AChE.(AU)

Kinetic and toxicological effects of synthesized palladium(II) complex on snake venom (Bungarus sindanus) acetylcholinesterase

The venom of the krait (Bungarus sindanus), an Elapidae snake, is highly toxic to humans and contains a great amount of acetylcholinesterase (AChE). The enzyme AChE provokes the hydrolysis of substrate acetylcholine (ACh) in the nervous system and terminates nerve impulse. Different inhibitors inactivate AChE and lead to ACh accumulation and disrupted neurotransmission. Methods: The present study was designed to evaluate the effect of palladium(II) complex as antivenom against krait venom AChE using kinetics methods. Results: Statistical analysis showed that krait venom AChE inhibition decreases with the increase of Pd(II) complex (0.025-0.05 µM) and exerted 61% inhibition against the AChE at a fixed concentration (0.5 mM) of ACh. Kinetic analysis using the Lineweaver Burk plot showed that Pd(II) caused a competitive inhibition. The compound Pd(II) complex binds at the active site of the enzyme. It was observed that K m (Michaelis-Menten constant of AChE-ACh into AChE and product) increased from 0.108 to 0.310 mM (45.74 to 318.35%) and V max remained constant with an increase of Pd(II) complex concentrations. In AChE K Iapp was found to increase from 0.0912 to 0.025 µM (29.82-72.58%) and did not affect the V maxapp with an increase of ACh from (0.05-1 mM). K i (inhibitory constant) was estimated to be 0.029µM for snake venom; while the K m was estimated to be 0.4 mM. The calculated IC50 for Pd(II) complex was found to be 0.043 µM at constant ACh concentration (0.5 mM). Conclusions: The results show that the Pd(II) complex can be deliberated as an inhibitor of AChE.(AU)

Varespladib (LY315920) inhibits neuromuscular blockade induced by Oxyuranus scutellatus venom in a nerve-muscle preparation.

The phospholipase A2 (PLA2) inhibitors varespladib (LY315920) and its orally available derivative methyl-varespladib (LY333013) have been proposed as potential therapies for the treatment of snakebite envenomings in which toxicity depends on the action of PLA2s. In this study, the ability of LY315920 to abrogate the effect of the potent neurotoxic venom of Oxyuranus scutellatus (taipan) was assessed using the mouse phrenic nerve-diaphragm preparation. LY315920 inhibited the venom when (a) incubated with venom before addition to the medium; (b) added to the medium before addition of venom, and; (c) added to the medium within 30 min after addition of venom, and even after the onset of decline in twitch response. This contrasts with previous results with antivenom using the same experimental model, in which the window of time when antibodies are effective is shorter than 10 min. It is proposed that such differences may depend either on the higher affinity of the inhibitor for PLA2s or on the possibility that LY315920 reaches the cytosol of the nerve terminals, inhibiting neurotoxins that have been internalized. Our findings bear implications on the therapeutic potential of varespladib in neurotoxic snakebite envenomings mediated by presynaptically-acting PLA2s.

Novel three-finger toxins from Micrurus dumerilii and Micrurus mipartitus coral snake venoms: Phylogenetic relationships and characterization of Clarkitoxin-I-Mdum.

Micrurus mipartitus and M. dumerilii are the most medically important coral snakes in Colombia. Proteomic characterization of their venoms has previously shown that proteins of the three-finger toxin (3FTx) family are abundant components, especially in M. mipartitus (61%) and to a lesser extent in M. dumerilii (28%). In order to increase knowledge on these toxins, in this work a major 3FTx of M. dumerilii venom (8% of the venom proteins), named Clarkitoxin-I-Mdum, was isolated and characterized. Its amino acid sequence comprises 66 residues, with an isotope-averaged molecular mass of 7537 ±â€¯2 Da and a theoretical pI of 9.36, presenting the conserved pattern of eight cysteines that classifies it as a short-chain (type I) 3FTx. Clarkitoxin-I-Mdum was not lethal to mice by intravenous or intracerebroventricular route and was not cytolytic to myogenic cells in vitro. On the other hand, five coding sequences for 3FTxs were obtained from the venom gland of M. mipartitus. These novel toxin sequences were named Mm3FTx-01 to Mm3FTx-05, all of them also presenting the eight conserved cysteines of short-chain 3FTxs. Phylogenetic analysis revealed high variability of 3FTxs from Micrurus, and ELISA using antibodies raised to the major 3FTxs from M. mipartitus and M. dumerilii confirmed their immunochemical divergence. These results highlight the relevance of performing further studies aiming at a deeper understanding of the functional and antigenic relationships among specific Micrurus toxins, with important implications for the production of antivenoms.