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Carlwilhelm Vogel - One of the best experts on this subject based on the ideXlab platform.

  • identification of intermolecular bonds between human factor b and Cobra Venom factor important for c3 convertase stability
    Toxicon, 2020
    Co-Authors: Brian E. Hew, Carlwilhelm Vogel, Michael K Pangburn, David C. Fritzinger
    Abstract:

    Abstract Cobra Venom factor (CVF) is the complement-activating protein in Cobra Venom. CVF is a structural and functional analog of complement component C3. CVF, like C3b, forms a convertase with factor B. This bimolecular complex CVF, Bb is an enzyme that cleaves C3 and C5. However, CVF, Bb exhibits significantly different functional properties from C3b,Bb. Whereas both, CVF, Bb and C3b, Bb exhibit spontaneous decay-dissociation into the respective subunits, thereby eliminating the enzymatic activity, the CVF, Bb convertase is physico-chemically far more stable, decaying with a half-life that is more than two orders of magnitude slower than that of C3b,Bb. In addition, CVF, Bb is completely resistant to inactivation by Factors H and I. These two properties of CVF, Bb allow continuous activation of C3 and C5, and complement depletion in serum. In order to understand the structural basis for the physico-chemical stability of CVF,Bb, we have created recombinant hybrid proteins of CVF and human C3, based on structural differences between CVF and human C3b in the C-terminal C345C domain. Here we describe three human C3/CVF hybrid proteins which differ in only one, two, or five amino acid residues from earlier described hybrid proteins. In all three cases, the hybrid proteins containing CVF residues form more stable convertases, and exhibit stronger complement-depletion activity than hybrid proteins with human C3 residues. Three bonds between CVF residues and Factor Bb residues could be identified by crystallographic modeling that contribute to the greater stability of the convertases.

  • Absence of a neutralizing antibody response to humanized Cobra Venom factor in mice
    Molecular Immunology, 2018
    Co-Authors: Mathieu Ing, Carlwilhelm Vogel, David C. Fritzinger, Brian Hew, Sandrine Delignat, Sébastien Lacroix-desmazes, Julie Rayes
    Abstract:

    Cobra Venom factor (CVF) is the complement-activating protein in Cobra Venom. Humanized CVF (hCVF) is a human C3 derivative where the C-terminal 168 amino acid residues were replaced with the homologous sequence from CVF. hCVF has been shown in multiple models of disease with complement pathology to be a promising therapeutic agent, with no observed adverse effects. Here we describe the antibody response to hCVF in two different strains of mice. hCVF was able to repeatedly decomplement the mice after four injections in weekly intervals, demonstrating the absence of a neutralizing antibody response. In contrast, natural CVF caused decomplementation in all mice only after the first administration. After two additional administrations of natural CVF, decomplementation was inconsistent and varied tremendously from mouse to mouse. After the fourth administration, natural CVF was essentially unable to deplete complement, consistent with the known generation of a neutralizing antibody response. We also analyzed the IgG antibody response to hCVF. There was great variation, with approximately one quarter of the mice exhibiting non-detectable levels of anti-hCVF IgG, and another quarter very low levels. The levels of anti-hCVF IgG did not correlate with the levels of remaining C3. The anti-hCVF antibodies cross-reacted with natural CVF, recombinant CVF, and human C3. Whereas overall the level of anti-hCVF IgG cross-reacting with human C3 was lower compared to rCVF or nCVF, mice with higher levels of anti-hCVF IgG exhibited higher binding to CVF and human C3, excluding the possibility that higher antibody levels reflect preferential immunogenicity of CVF-specific or human C3-specific epitopes.

  • humanized Cobra Venom factor structure activity and therapeutic efficacy in preclinical disease models
    Molecular Immunology, 2014
    Co-Authors: Carlwilhelm Vogel, Paul W Finnegan, David C. Fritzinger
    Abstract:

    The complement system is an integral component of both innate and adaptive immunity. However, complement is also a pathogenetic factor in many diseases. The development of agents for therapeutic complement inhibition is the topic of intense investigations by many investigators. We have developed a distinctly different therapeutic approach: complement depletion rather than inhibition. This approach is based on Cobra Venom factor (CVF), a C3 analog known to be able to safely deplete complement. This manuscript will briefly review the structure and activity of CVF, along with its similarities and differences to C3. Exploiting the knowledge of the structure/function relationship of CVF and C3, we created derivatives of human C3 which display the CVF-like activity of depleting complement, referred to as humanized CVF (hCVF). This review describes the structure and activity of hCVF, including the important property of not cleaving C5. The efficacy of hCVF for therapeutic complement depletion in nine preclinical models diseases with complement pathology is reviewed, including reperfusion injury, age-related macular degeneration (AMD), paroxysmal nocturnal hemoglobinuria (PNH), and immunogenicity of Factor VIII in hemophilia A. Complement depletion is characterized by the absence of toxicity, even after intra-arterial injection into the pulmonary artery of primates. No immunogenicity has been observed.

  • complement depletion with humanised Cobra Venom factor efficacy in preclinical models of vascular diseases
    Thrombosis and Haemostasis, 2014
    Co-Authors: Carlwilhelm Vogel, David C. Fritzinger, William B Gorsuch, Gregory L Stahl
    Abstract:

    The complement system is an intrinsic part of the immune system and has important functions in both innate and adaptive immunity. On the other hand, inadvertent or misdirected complement activation is also involved in the pathogenesis of many diseases, contributing solely or significantly to tissue injury and disease development. Multiple approaches to develop pharmacological agents to inhibit complement are currently being pursued. We have developed a conceptually different approach of not inhibiting but depleting complement, based on the complement-depleting activities of Cobra Venom factor (CVF), a non-toxic Cobra Venom component with structural and functional homology to complement component C3. We developed a humanised version of CVF by creating human complement component C3 derivatives with complement-depleting activities of CVF (humanised CVF) as a promising therapeutic agent for diseases with complement pathogenesis. Here we review the beneficial therapeutic effect of humanised CVF in several murine models of vascular diseases such as reperfusion injury.

  • Cobra Venom factor structure function and humanization for therapeutic complement depletion
    Toxicon, 2010
    Co-Authors: Carlwilhelm Vogel, David C. Fritzinger
    Abstract:

    Cobra Venom factor (CVF) is the complement-activating protein in Cobra Venom. This manuscript reviews the structure and function of CVF, how it interacts with the complement system, the structural and functional homology to complement component C3, and the use of CVF as an experimental tool to decomplement laboratory animals to study the functions of complement in host defense and immune response as well as in the pathogenesis of diseases. This manuscript also reviews the recent progress in using the homology between CVF and C3 to study C3 structure and function, and to develop human C3 derivatives with the complement-depleting function of CVF. These human C3 derivatives represent humanized CVF, and are a conceptually different concept for pharmacological intervention of the complement system, therapeutic complement depletion. The use of humanized CVF for therapeutic complement depletion in several pre-clinical models of human diseases is also reviewed.

David C. Fritzinger - One of the best experts on this subject based on the ideXlab platform.

  • identification of intermolecular bonds between human factor b and Cobra Venom factor important for c3 convertase stability
    Toxicon, 2020
    Co-Authors: Brian E. Hew, Carlwilhelm Vogel, Michael K Pangburn, David C. Fritzinger
    Abstract:

    Abstract Cobra Venom factor (CVF) is the complement-activating protein in Cobra Venom. CVF is a structural and functional analog of complement component C3. CVF, like C3b, forms a convertase with factor B. This bimolecular complex CVF, Bb is an enzyme that cleaves C3 and C5. However, CVF, Bb exhibits significantly different functional properties from C3b,Bb. Whereas both, CVF, Bb and C3b, Bb exhibit spontaneous decay-dissociation into the respective subunits, thereby eliminating the enzymatic activity, the CVF, Bb convertase is physico-chemically far more stable, decaying with a half-life that is more than two orders of magnitude slower than that of C3b,Bb. In addition, CVF, Bb is completely resistant to inactivation by Factors H and I. These two properties of CVF, Bb allow continuous activation of C3 and C5, and complement depletion in serum. In order to understand the structural basis for the physico-chemical stability of CVF,Bb, we have created recombinant hybrid proteins of CVF and human C3, based on structural differences between CVF and human C3b in the C-terminal C345C domain. Here we describe three human C3/CVF hybrid proteins which differ in only one, two, or five amino acid residues from earlier described hybrid proteins. In all three cases, the hybrid proteins containing CVF residues form more stable convertases, and exhibit stronger complement-depletion activity than hybrid proteins with human C3 residues. Three bonds between CVF residues and Factor Bb residues could be identified by crystallographic modeling that contribute to the greater stability of the convertases.

  • Absence of a neutralizing antibody response to humanized Cobra Venom factor in mice
    Molecular Immunology, 2018
    Co-Authors: Mathieu Ing, Carlwilhelm Vogel, David C. Fritzinger, Brian Hew, Sandrine Delignat, Sébastien Lacroix-desmazes, Julie Rayes
    Abstract:

    Cobra Venom factor (CVF) is the complement-activating protein in Cobra Venom. Humanized CVF (hCVF) is a human C3 derivative where the C-terminal 168 amino acid residues were replaced with the homologous sequence from CVF. hCVF has been shown in multiple models of disease with complement pathology to be a promising therapeutic agent, with no observed adverse effects. Here we describe the antibody response to hCVF in two different strains of mice. hCVF was able to repeatedly decomplement the mice after four injections in weekly intervals, demonstrating the absence of a neutralizing antibody response. In contrast, natural CVF caused decomplementation in all mice only after the first administration. After two additional administrations of natural CVF, decomplementation was inconsistent and varied tremendously from mouse to mouse. After the fourth administration, natural CVF was essentially unable to deplete complement, consistent with the known generation of a neutralizing antibody response. We also analyzed the IgG antibody response to hCVF. There was great variation, with approximately one quarter of the mice exhibiting non-detectable levels of anti-hCVF IgG, and another quarter very low levels. The levels of anti-hCVF IgG did not correlate with the levels of remaining C3. The anti-hCVF antibodies cross-reacted with natural CVF, recombinant CVF, and human C3. Whereas overall the level of anti-hCVF IgG cross-reacting with human C3 was lower compared to rCVF or nCVF, mice with higher levels of anti-hCVF IgG exhibited higher binding to CVF and human C3, excluding the possibility that higher antibody levels reflect preferential immunogenicity of CVF-specific or human C3-specific epitopes.

  • humanized Cobra Venom factor structure activity and therapeutic efficacy in preclinical disease models
    Molecular Immunology, 2014
    Co-Authors: Carlwilhelm Vogel, Paul W Finnegan, David C. Fritzinger
    Abstract:

    The complement system is an integral component of both innate and adaptive immunity. However, complement is also a pathogenetic factor in many diseases. The development of agents for therapeutic complement inhibition is the topic of intense investigations by many investigators. We have developed a distinctly different therapeutic approach: complement depletion rather than inhibition. This approach is based on Cobra Venom factor (CVF), a C3 analog known to be able to safely deplete complement. This manuscript will briefly review the structure and activity of CVF, along with its similarities and differences to C3. Exploiting the knowledge of the structure/function relationship of CVF and C3, we created derivatives of human C3 which display the CVF-like activity of depleting complement, referred to as humanized CVF (hCVF). This review describes the structure and activity of hCVF, including the important property of not cleaving C5. The efficacy of hCVF for therapeutic complement depletion in nine preclinical models diseases with complement pathology is reviewed, including reperfusion injury, age-related macular degeneration (AMD), paroxysmal nocturnal hemoglobinuria (PNH), and immunogenicity of Factor VIII in hemophilia A. Complement depletion is characterized by the absence of toxicity, even after intra-arterial injection into the pulmonary artery of primates. No immunogenicity has been observed.

  • complement depletion with humanised Cobra Venom factor efficacy in preclinical models of vascular diseases
    Thrombosis and Haemostasis, 2014
    Co-Authors: Carlwilhelm Vogel, David C. Fritzinger, William B Gorsuch, Gregory L Stahl
    Abstract:

    The complement system is an intrinsic part of the immune system and has important functions in both innate and adaptive immunity. On the other hand, inadvertent or misdirected complement activation is also involved in the pathogenesis of many diseases, contributing solely or significantly to tissue injury and disease development. Multiple approaches to develop pharmacological agents to inhibit complement are currently being pursued. We have developed a conceptually different approach of not inhibiting but depleting complement, based on the complement-depleting activities of Cobra Venom factor (CVF), a non-toxic Cobra Venom component with structural and functional homology to complement component C3. We developed a humanised version of CVF by creating human complement component C3 derivatives with complement-depleting activities of CVF (humanised CVF) as a promising therapeutic agent for diseases with complement pathogenesis. Here we review the beneficial therapeutic effect of humanised CVF in several murine models of vascular diseases such as reperfusion injury.

  • Cobra Venom factor structure function and humanization for therapeutic complement depletion
    Toxicon, 2010
    Co-Authors: Carlwilhelm Vogel, David C. Fritzinger
    Abstract:

    Cobra Venom factor (CVF) is the complement-activating protein in Cobra Venom. This manuscript reviews the structure and function of CVF, how it interacts with the complement system, the structural and functional homology to complement component C3, and the use of CVF as an experimental tool to decomplement laboratory animals to study the functions of complement in host defense and immune response as well as in the pathogenesis of diseases. This manuscript also reviews the recent progress in using the homology between CVF and C3 to study C3 structure and function, and to develop human C3 derivatives with the complement-depleting function of CVF. These human C3 derivatives represent humanized CVF, and are a conceptually different concept for pharmacological intervention of the complement system, therapeutic complement depletion. The use of humanized CVF for therapeutic complement depletion in several pre-clinical models of human diseases is also reviewed.

Nget Hong Tan - One of the best experts on this subject based on the ideXlab platform.

  • immunological cross reactivity and neutralization of the principal toxins of naja sumatrana and related Cobra Venoms by a thai polyvalent antiVenom neuro polyvalent snake antiVenom
    Acta Tropica, 2015
    Co-Authors: Poh Kuan Leong, Si Mui Sim, Shin Yee Fung, Choo Hock Tan, Nget Hong Tan
    Abstract:

    The low potency of Cobra antiVenom has been an area of concern in immunotherapy for Cobra enVenomation. This study sought to investigate factors limiting the neutralizing potency of Cobra antiVenom, using a murine model. We examined the immunological reactivity and neutralizing potency of a Thai polyvalent antiVenom against the principal toxins of Naja sumatrana (Equatorial spitting Cobra) Venom and two related Asiatic Cobra Venom -neurotoxins. The antiVenom possesses moderate neutralizing potency against phospholipases A2 (P, potency of 0.98 mg/mL) and moderately weak neutralizing potency against long-chain -neurotoxins (0.26–0.42 mg/mL) but was only weakly effective in neutralizing the short-chain -neurotoxins and cardiotoxins (0.05–0.08 mg/mL). The poor neutralizing potency of the antiVenom on the low molecular mass short-chain neurotoxins and cardiotoxins is presumably the main limiting factor of the efficacy of the Cobra antiVenom. Our results also showed that phospholipase A2, which exhibited the highest ELISA reactivity and avidity, was most effectively neutralized, whereas N. sumatrana short-chain neurotoxin, which exhibited the lowest ELISA reactivity and avidity, was least effectively neutralized by the antiVenom. These observations suggest that low immunoreactivity (low ELISA reactivity and avidity) is one of the reasons for poor neutralization of the Cobra Venom low molecular mass toxins. Nevertheless, the overall results show that there is a lack of congruence between the immunological reactivity of the toxins toward antiVenom and the effectiveness of toxin neutralization by the antiVenom, indicating that there are other factors that also contribute to the weak neutralization capacity of the antiVenom. Several suggestions have been put forward to overcome the low efficacy of the Cobra antiVenom. The use of a ‘proper-mix’ formulation of Cobra Venoms as immunogen, whereby the immunogen mixture used for hyperimmunization contains a mix of various types of -neurotoxins and cardiotoxins in sufficient amount, may also help to improve the efficacy and broaden the neutralization spectrum of the antiVenom. © 2015 Elsevier B.V. All rights reserved.

  • pharmacokinetics of naja sumatrana equatorial spitting Cobra Venom and its major toxins in experimentally enVenomed rabbits
    PLOS Neglected Tropical Diseases, 2014
    Co-Authors: Michelle Khai Khun Yap, Nget Hong Tan, Shin Yee Fung, Si Mui Sim, Choo Hock Tan
    Abstract:

    Background The optimization of snakebite management and the use of antiVenom depend greatly on the knowledge of the Venom's composition as well as its pharmacokinetics. To date, however, pharmacokinetic reports on Cobra Venoms and their toxins are still relatively limited. In the present study, we investigated the pharmacokinetics of Naja sumatrana (Equatorial spitting Cobra) Venom and its major toxins (phospholipase A2, neurotoxin and cardiotoxin), following intravenous and intramuscular administration into rabbits. Principal findings The serum antigen concentration-time profile of the N. sumatrana Venom and its major toxins injected intravenously fitted a two-compartment model of pharmacokinetics. The systemic clearance (91.3 ml/h), terminal phase half-life (13.6 h) and systemic bioavailability (41.9%) of N. sumatrana Venom injected intramuscularly were similar to those of N. sputatrix Venom determined in an earlier study. The Venom neurotoxin and cardiotoxin reached their peak concentrations within 30 min following intramuscular injection, relatively faster than the phospholipase A2 and whole Venom (Tmax = 2 h and 1 h, respectively). Rapid absorption of the neurotoxin and cardiotoxin from the injection site into systemic circulation indicates fast onsets of action of these principal toxins that are responsible for the early systemic manifestation of enVenoming. The more prominent role of the neurotoxin in N. sumatrana systemic enVenoming is further supported by its significantly higher intramuscular bioavailability (Fi.m. = 81.5%) compared to that of the phospholipase A2 (Fi.m. = 68.6%) or cardiotoxin (Fi.m. = 45.6%). The incomplete absorption of the phospholipase A2 and cardiotoxin may infer the toxins' affinities for tissues at the injection site and their pathological roles in local tissue damages through synergistic interactions. Conclusion/Significance Our results suggest that the Venom neurotoxin is absorbed very rapidly and has the highest bioavailability following intramuscular injection, supporting its role as the principal toxin in systemic enVenoming.

  • proteomic characterization of Venom of the medically important southeast asian naja sumatrana equatorial spitting Cobra
    Acta Tropica, 2014
    Co-Authors: Michelle Khai Khun Yap, Shin Yee Fung, Kae Yi Tan, Nget Hong Tan
    Abstract:

    The proteome of Naja sumatrana (Equatorial spitting Cobra) Venom was investigated by shotgun analysis and a combination of ion-exchange chromatography and reverse phase HPLC. Shotgun analysis revealed the presence of 39 proteins in the Venom while the chromatographic approach identified 37 Venom proteins. The results indicated that, like other Asiatic Cobra Venoms, N. sumatrana contains large number of three finger toxins and phospholipases A2, which together constitute 92.1% by weight of Venom protein. However, only eight of the toxins can be considered as major Venom toxins. These include two phospholipases A2, three neurotoxins (two long neurotoxins and a short neurotoxin) and three cardiotoxins. The eight major toxins have relative abundance of 1.6–27.2% Venom proteins and together account for 89.8% (by weight) of total Venom protein. Other Venom proteins identified include Zn-metalloproteinase-disintegrin, Thaicobrin, CRISP, natriuretic peptide, complement depleting factors, Cobra Venom factors, Venom nerve growth factor and Cobra serum albumin. The proteome of N. sumatrana Venom is similar to proteome of other Asiatic Cobra Venoms but differs from that of African spitting Cobra Venom. Our results confirm that the main toxic action of N. sumatrana Venom is neurotoxic but the large amount of cardiotoxins and phospholipases A2 are likely to contribute significantly to the overall pathophysiological action of the Venom. The differences in toxin distribution between N. sumatrana Venom and African spitting Cobra Venoms suggest possible differences in the pathophysiological actions of N. sumatrana Venom and the African spitting Cobra Venoms, and explain why antiVenom raised against Asiatic Cobra Venom is not effective against African spitting Cobra Venoms.

  • Antiproliferative Activity of King Cobra (Ophiophagus hannah) Venom l-Amino Acid Oxidase
    Basic & clinical pharmacology & toxicology, 2013
    Co-Authors: Mui Li Lee, Shin Yee Fung, Ivy Chung, M.s. Kanthimathi, Nget Hong Tan
    Abstract:

    King Cobra (Ophiophagus hannah) Venom l-amino acid oxidase (LAAO), a heat-stable enzyme, is an extremely potent antiproliferative agent against cancer cells when compared with LAAO isolated from other snake Venoms. King Cobra Venom LAAO was shown to exhibit very strong antiproliferative activities against MCF-7 (human breast adenocarcinoma) and A549 (human lung adenocarcinoma) cells, with an IC50 value of 0.04 ± 0.00 and 0.05 ± 0.00 μg/mL, respectively, after 72-hr treatment. In comparison, its cytotoxicity was about 3–4 times lower when tested against human non-tumourigenic breast (184B5) and lung (NL 20) cells, suggesting selective antitumour activity. Furthermore, its potency in MCF-7 and A549 cell lines was greater than the effects of doxorubicin, a clinically established cancer chemotherapeutic agent, which showed an IC50 value of 0.18 ± 0.03 and 0.63 ± 0.21 μg/mL, respectively, against the two cell lines. The selective cytotoxic action of the LAAO was confirmed by phycoerythrin (PE) annexin V/7-amino-actinomycin (AAD) apoptotic assay, in which a significant increase in apoptotic cells was observed in LAAO-treated tumour cells than in their non-tumourigenic counterparts. The ability of LAAO to induce apoptosis in tumour cells was further demonstrated using caspase-3/7 and DNA fragmentation assays. We also determined that this enzyme may target oxidative stress in its killing of tumour cells, as its cytotoxicity was significantly reduced in the presence of catalase (a H2O2 scavenger). In view of its heat stability and selective and potent cytotoxic action on cancer cells, king Cobra Venom LAAO can be potentially developed for treating solid tumours.

  • toxicokinetics of naja sputatrix javan spitting Cobra Venom following intramuscular and intravenous administrations of the Venom into rabbits
    Toxicon, 2013
    Co-Authors: Michelle Khai Khun Yap, Nget Hong Tan, Si Mui Sim, Shin Yee Fung
    Abstract:

    Existing protocols for antiVenom treatment of snake enVenomations are generally not well optimized due partly to inadequate knowledge of the toxicokinetics of Venoms. The toxicokinetics of Naja sputatrix (Javan spitting Cobra) Venom was investigated following intravenous and intramuscular injections of the Venom into rabbits using doublesandwich ELISA. The toxicokinetics of the Venom injected intravenously fitted a twocompartment model. When the Venom was injected intramuscularly, the serum concentration–time profile exhibited a more complex absorption and/or distribution pattern. Nevertheless, the terminal half-life, volume of distribution by area and systemic clearance of the Venom injected intramuscularly were not significantly different (p > 0.05) from that of the Venom injected intravenously. The systemic bioavailability of the Venom antigens injected by intramuscular route was 41.7%. Our toxicokinetic finding is consistent with other reports, and may indicate that some Cobra Venom toxins have high affinity for the tissues at the site of injection. Our results suggest that the intramuscular route of administration doesn’t significantly alter the toxicokinetics of N. sputatrix Venom although it significantly reduces the systemic bioavailability of the Venom.

Shin Yee Fung - One of the best experts on this subject based on the ideXlab platform.

  • immunological cross reactivity and neutralization of the principal toxins of naja sumatrana and related Cobra Venoms by a thai polyvalent antiVenom neuro polyvalent snake antiVenom
    Acta Tropica, 2015
    Co-Authors: Poh Kuan Leong, Si Mui Sim, Shin Yee Fung, Choo Hock Tan, Nget Hong Tan
    Abstract:

    The low potency of Cobra antiVenom has been an area of concern in immunotherapy for Cobra enVenomation. This study sought to investigate factors limiting the neutralizing potency of Cobra antiVenom, using a murine model. We examined the immunological reactivity and neutralizing potency of a Thai polyvalent antiVenom against the principal toxins of Naja sumatrana (Equatorial spitting Cobra) Venom and two related Asiatic Cobra Venom -neurotoxins. The antiVenom possesses moderate neutralizing potency against phospholipases A2 (P, potency of 0.98 mg/mL) and moderately weak neutralizing potency against long-chain -neurotoxins (0.26–0.42 mg/mL) but was only weakly effective in neutralizing the short-chain -neurotoxins and cardiotoxins (0.05–0.08 mg/mL). The poor neutralizing potency of the antiVenom on the low molecular mass short-chain neurotoxins and cardiotoxins is presumably the main limiting factor of the efficacy of the Cobra antiVenom. Our results also showed that phospholipase A2, which exhibited the highest ELISA reactivity and avidity, was most effectively neutralized, whereas N. sumatrana short-chain neurotoxin, which exhibited the lowest ELISA reactivity and avidity, was least effectively neutralized by the antiVenom. These observations suggest that low immunoreactivity (low ELISA reactivity and avidity) is one of the reasons for poor neutralization of the Cobra Venom low molecular mass toxins. Nevertheless, the overall results show that there is a lack of congruence between the immunological reactivity of the toxins toward antiVenom and the effectiveness of toxin neutralization by the antiVenom, indicating that there are other factors that also contribute to the weak neutralization capacity of the antiVenom. Several suggestions have been put forward to overcome the low efficacy of the Cobra antiVenom. The use of a ‘proper-mix’ formulation of Cobra Venoms as immunogen, whereby the immunogen mixture used for hyperimmunization contains a mix of various types of -neurotoxins and cardiotoxins in sufficient amount, may also help to improve the efficacy and broaden the neutralization spectrum of the antiVenom. © 2015 Elsevier B.V. All rights reserved.

  • pharmacokinetics of naja sumatrana equatorial spitting Cobra Venom and its major toxins in experimentally enVenomed rabbits
    PLOS Neglected Tropical Diseases, 2014
    Co-Authors: Michelle Khai Khun Yap, Nget Hong Tan, Shin Yee Fung, Si Mui Sim, Choo Hock Tan
    Abstract:

    Background The optimization of snakebite management and the use of antiVenom depend greatly on the knowledge of the Venom's composition as well as its pharmacokinetics. To date, however, pharmacokinetic reports on Cobra Venoms and their toxins are still relatively limited. In the present study, we investigated the pharmacokinetics of Naja sumatrana (Equatorial spitting Cobra) Venom and its major toxins (phospholipase A2, neurotoxin and cardiotoxin), following intravenous and intramuscular administration into rabbits. Principal findings The serum antigen concentration-time profile of the N. sumatrana Venom and its major toxins injected intravenously fitted a two-compartment model of pharmacokinetics. The systemic clearance (91.3 ml/h), terminal phase half-life (13.6 h) and systemic bioavailability (41.9%) of N. sumatrana Venom injected intramuscularly were similar to those of N. sputatrix Venom determined in an earlier study. The Venom neurotoxin and cardiotoxin reached their peak concentrations within 30 min following intramuscular injection, relatively faster than the phospholipase A2 and whole Venom (Tmax = 2 h and 1 h, respectively). Rapid absorption of the neurotoxin and cardiotoxin from the injection site into systemic circulation indicates fast onsets of action of these principal toxins that are responsible for the early systemic manifestation of enVenoming. The more prominent role of the neurotoxin in N. sumatrana systemic enVenoming is further supported by its significantly higher intramuscular bioavailability (Fi.m. = 81.5%) compared to that of the phospholipase A2 (Fi.m. = 68.6%) or cardiotoxin (Fi.m. = 45.6%). The incomplete absorption of the phospholipase A2 and cardiotoxin may infer the toxins' affinities for tissues at the injection site and their pathological roles in local tissue damages through synergistic interactions. Conclusion/Significance Our results suggest that the Venom neurotoxin is absorbed very rapidly and has the highest bioavailability following intramuscular injection, supporting its role as the principal toxin in systemic enVenoming.

  • proteomic characterization of Venom of the medically important southeast asian naja sumatrana equatorial spitting Cobra
    Acta Tropica, 2014
    Co-Authors: Michelle Khai Khun Yap, Shin Yee Fung, Kae Yi Tan, Nget Hong Tan
    Abstract:

    The proteome of Naja sumatrana (Equatorial spitting Cobra) Venom was investigated by shotgun analysis and a combination of ion-exchange chromatography and reverse phase HPLC. Shotgun analysis revealed the presence of 39 proteins in the Venom while the chromatographic approach identified 37 Venom proteins. The results indicated that, like other Asiatic Cobra Venoms, N. sumatrana contains large number of three finger toxins and phospholipases A2, which together constitute 92.1% by weight of Venom protein. However, only eight of the toxins can be considered as major Venom toxins. These include two phospholipases A2, three neurotoxins (two long neurotoxins and a short neurotoxin) and three cardiotoxins. The eight major toxins have relative abundance of 1.6–27.2% Venom proteins and together account for 89.8% (by weight) of total Venom protein. Other Venom proteins identified include Zn-metalloproteinase-disintegrin, Thaicobrin, CRISP, natriuretic peptide, complement depleting factors, Cobra Venom factors, Venom nerve growth factor and Cobra serum albumin. The proteome of N. sumatrana Venom is similar to proteome of other Asiatic Cobra Venoms but differs from that of African spitting Cobra Venom. Our results confirm that the main toxic action of N. sumatrana Venom is neurotoxic but the large amount of cardiotoxins and phospholipases A2 are likely to contribute significantly to the overall pathophysiological action of the Venom. The differences in toxin distribution between N. sumatrana Venom and African spitting Cobra Venoms suggest possible differences in the pathophysiological actions of N. sumatrana Venom and the African spitting Cobra Venoms, and explain why antiVenom raised against Asiatic Cobra Venom is not effective against African spitting Cobra Venoms.

  • Antiproliferative Activity of King Cobra (Ophiophagus hannah) Venom l-Amino Acid Oxidase
    Basic & clinical pharmacology & toxicology, 2013
    Co-Authors: Mui Li Lee, Shin Yee Fung, Ivy Chung, M.s. Kanthimathi, Nget Hong Tan
    Abstract:

    King Cobra (Ophiophagus hannah) Venom l-amino acid oxidase (LAAO), a heat-stable enzyme, is an extremely potent antiproliferative agent against cancer cells when compared with LAAO isolated from other snake Venoms. King Cobra Venom LAAO was shown to exhibit very strong antiproliferative activities against MCF-7 (human breast adenocarcinoma) and A549 (human lung adenocarcinoma) cells, with an IC50 value of 0.04 ± 0.00 and 0.05 ± 0.00 μg/mL, respectively, after 72-hr treatment. In comparison, its cytotoxicity was about 3–4 times lower when tested against human non-tumourigenic breast (184B5) and lung (NL 20) cells, suggesting selective antitumour activity. Furthermore, its potency in MCF-7 and A549 cell lines was greater than the effects of doxorubicin, a clinically established cancer chemotherapeutic agent, which showed an IC50 value of 0.18 ± 0.03 and 0.63 ± 0.21 μg/mL, respectively, against the two cell lines. The selective cytotoxic action of the LAAO was confirmed by phycoerythrin (PE) annexin V/7-amino-actinomycin (AAD) apoptotic assay, in which a significant increase in apoptotic cells was observed in LAAO-treated tumour cells than in their non-tumourigenic counterparts. The ability of LAAO to induce apoptosis in tumour cells was further demonstrated using caspase-3/7 and DNA fragmentation assays. We also determined that this enzyme may target oxidative stress in its killing of tumour cells, as its cytotoxicity was significantly reduced in the presence of catalase (a H2O2 scavenger). In view of its heat stability and selective and potent cytotoxic action on cancer cells, king Cobra Venom LAAO can be potentially developed for treating solid tumours.

  • toxicokinetics of naja sputatrix javan spitting Cobra Venom following intramuscular and intravenous administrations of the Venom into rabbits
    Toxicon, 2013
    Co-Authors: Michelle Khai Khun Yap, Nget Hong Tan, Si Mui Sim, Shin Yee Fung
    Abstract:

    Existing protocols for antiVenom treatment of snake enVenomations are generally not well optimized due partly to inadequate knowledge of the toxicokinetics of Venoms. The toxicokinetics of Naja sputatrix (Javan spitting Cobra) Venom was investigated following intravenous and intramuscular injections of the Venom into rabbits using doublesandwich ELISA. The toxicokinetics of the Venom injected intravenously fitted a twocompartment model. When the Venom was injected intramuscularly, the serum concentration–time profile exhibited a more complex absorption and/or distribution pattern. Nevertheless, the terminal half-life, volume of distribution by area and systemic clearance of the Venom injected intramuscularly were not significantly different (p > 0.05) from that of the Venom injected intravenously. The systemic bioavailability of the Venom antigens injected by intramuscular route was 41.7%. Our toxicokinetic finding is consistent with other reports, and may indicate that some Cobra Venom toxins have high affinity for the tissues at the site of injection. Our results suggest that the intramuscular route of administration doesn’t significantly alter the toxicokinetics of N. sputatrix Venom although it significantly reduces the systemic bioavailability of the Venom.

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  • pharmacokinetics of naja sumatrana equatorial spitting Cobra Venom and its major toxins in experimentally enVenomed rabbits
    PLOS Neglected Tropical Diseases, 2014
    Co-Authors: Michelle Khai Khun Yap, Nget Hong Tan, Shin Yee Fung, Si Mui Sim, Choo Hock Tan
    Abstract:

    Background The optimization of snakebite management and the use of antiVenom depend greatly on the knowledge of the Venom's composition as well as its pharmacokinetics. To date, however, pharmacokinetic reports on Cobra Venoms and their toxins are still relatively limited. In the present study, we investigated the pharmacokinetics of Naja sumatrana (Equatorial spitting Cobra) Venom and its major toxins (phospholipase A2, neurotoxin and cardiotoxin), following intravenous and intramuscular administration into rabbits. Principal findings The serum antigen concentration-time profile of the N. sumatrana Venom and its major toxins injected intravenously fitted a two-compartment model of pharmacokinetics. The systemic clearance (91.3 ml/h), terminal phase half-life (13.6 h) and systemic bioavailability (41.9%) of N. sumatrana Venom injected intramuscularly were similar to those of N. sputatrix Venom determined in an earlier study. The Venom neurotoxin and cardiotoxin reached their peak concentrations within 30 min following intramuscular injection, relatively faster than the phospholipase A2 and whole Venom (Tmax = 2 h and 1 h, respectively). Rapid absorption of the neurotoxin and cardiotoxin from the injection site into systemic circulation indicates fast onsets of action of these principal toxins that are responsible for the early systemic manifestation of enVenoming. The more prominent role of the neurotoxin in N. sumatrana systemic enVenoming is further supported by its significantly higher intramuscular bioavailability (Fi.m. = 81.5%) compared to that of the phospholipase A2 (Fi.m. = 68.6%) or cardiotoxin (Fi.m. = 45.6%). The incomplete absorption of the phospholipase A2 and cardiotoxin may infer the toxins' affinities for tissues at the injection site and their pathological roles in local tissue damages through synergistic interactions. Conclusion/Significance Our results suggest that the Venom neurotoxin is absorbed very rapidly and has the highest bioavailability following intramuscular injection, supporting its role as the principal toxin in systemic enVenoming.

  • proteomic characterization of Venom of the medically important southeast asian naja sumatrana equatorial spitting Cobra
    Acta Tropica, 2014
    Co-Authors: Michelle Khai Khun Yap, Shin Yee Fung, Kae Yi Tan, Nget Hong Tan
    Abstract:

    The proteome of Naja sumatrana (Equatorial spitting Cobra) Venom was investigated by shotgun analysis and a combination of ion-exchange chromatography and reverse phase HPLC. Shotgun analysis revealed the presence of 39 proteins in the Venom while the chromatographic approach identified 37 Venom proteins. The results indicated that, like other Asiatic Cobra Venoms, N. sumatrana contains large number of three finger toxins and phospholipases A2, which together constitute 92.1% by weight of Venom protein. However, only eight of the toxins can be considered as major Venom toxins. These include two phospholipases A2, three neurotoxins (two long neurotoxins and a short neurotoxin) and three cardiotoxins. The eight major toxins have relative abundance of 1.6–27.2% Venom proteins and together account for 89.8% (by weight) of total Venom protein. Other Venom proteins identified include Zn-metalloproteinase-disintegrin, Thaicobrin, CRISP, natriuretic peptide, complement depleting factors, Cobra Venom factors, Venom nerve growth factor and Cobra serum albumin. The proteome of N. sumatrana Venom is similar to proteome of other Asiatic Cobra Venoms but differs from that of African spitting Cobra Venom. Our results confirm that the main toxic action of N. sumatrana Venom is neurotoxic but the large amount of cardiotoxins and phospholipases A2 are likely to contribute significantly to the overall pathophysiological action of the Venom. The differences in toxin distribution between N. sumatrana Venom and African spitting Cobra Venoms suggest possible differences in the pathophysiological actions of N. sumatrana Venom and the African spitting Cobra Venoms, and explain why antiVenom raised against Asiatic Cobra Venom is not effective against African spitting Cobra Venoms.

  • toxicokinetics of naja sputatrix javan spitting Cobra Venom following intramuscular and intravenous administrations of the Venom into rabbits
    Toxicon, 2013
    Co-Authors: Michelle Khai Khun Yap, Nget Hong Tan, Si Mui Sim, Shin Yee Fung
    Abstract:

    Existing protocols for antiVenom treatment of snake enVenomations are generally not well optimized due partly to inadequate knowledge of the toxicokinetics of Venoms. The toxicokinetics of Naja sputatrix (Javan spitting Cobra) Venom was investigated following intravenous and intramuscular injections of the Venom into rabbits using doublesandwich ELISA. The toxicokinetics of the Venom injected intravenously fitted a twocompartment model. When the Venom was injected intramuscularly, the serum concentration–time profile exhibited a more complex absorption and/or distribution pattern. Nevertheless, the terminal half-life, volume of distribution by area and systemic clearance of the Venom injected intramuscularly were not significantly different (p > 0.05) from that of the Venom injected intravenously. The systemic bioavailability of the Venom antigens injected by intramuscular route was 41.7%. Our toxicokinetic finding is consistent with other reports, and may indicate that some Cobra Venom toxins have high affinity for the tissues at the site of injection. Our results suggest that the intramuscular route of administration doesn’t significantly alter the toxicokinetics of N. sputatrix Venom although it significantly reduces the systemic bioavailability of the Venom.