Pharmacokinetics of the Sri Lankan hump-nosed pit viper (Hypnale hypnale) venom following intravenous and intramuscular injections of the venom into rabbits.

The knowledge of venom pharmacokinetics is essential to improve the understanding of envenomation pathophysiology. Using a double-sandwich ELISA, this study investigated the pharmacokinetics of the venom of hump-nosed pit viper (Hypnale hypnale) following intravenous and intramuscular injections into rabbits. The pharmacokinetics of the venom injected intravenously fitted a three-compartment model. There is a rapid (t1/2π = 0.4 h) and a slow (t1/2α = 0.8 h) distribution phase, followed by a long elimination phase (t1/2ß = 19.3 h) with a systemic clearance of 6.8 mL h(-1) kg(-1), consistent with the prolonged abnormal hemostasis reported in H. hypnale envenomation. On intramuscular route, multiple peak concentrations observed in the beginning implied a more complex venom absorption and/or distribution pattern. The terminal half-life, volume of distribution by area and systemic clearance of the venom injected intramuscularly were nevertheless not significantly different (p > 0.05) from that of the venom injected intravenously. The intramuscular bioavailability was exceptionally low (Fi.m. = 4%), accountable for the highly varied median lethal doses between intravenous and intramuscular envenomations in animals. The findings indicate that the intramuscular route of administration does not significantly alter the pharmacokinetics of H. hypnale venom although it significantly reduces the systemic bioavailability of the venom.

A high affinity acceptor for phospholipase A2 with neurotoxic activity is a calmodulin.

One of the high affinity binding proteins for ammodytoxin C, a snake venom presynaptically neurotoxic phospholipase A(2), has been purified from porcine cerebral cortex and characterized. After extraction from the membranes, the toxin-binding protein was isolated in a homogenous form using wheat germ lectin-Sepharose, Q-Sepharose, and ammodytoxin-CH-Sepharose chromatography. The specific binding of (125)I-ammodytoxin C to the isolated acceptor was inhibited to different extents by some neurotoxic phospholipases A(2), ammodytoxins, bee venom phospholipase A(2), agkistrodotoxin, and crotoxin; but not by nontoxic phospholipases A(2), ammodytin I(2), porcine pancreatic phospholipase A(2), and human type IIA phospholipase A(2); suggesting the significance of the acceptor in the mechanism of phospholipase A(2) neurotoxicity. The isolated acceptor was identified as calmodulin by tandem mass spectrometry. Since calmodulin is generally considered as an intracellular protein, the identity of this acceptor supports the view that secretory phospholipase A(2) neurotoxins have to be internalized to exert their toxic effect. Moreover, since ammodytoxin is known to block synaptic transmission, its interaction with calmodulin as an acceptor may constitute a valuable probe for further investigation of the role of the latter in this Ca(2+)-regulated process.

A new monospecific ovine Fab fragment antivenom for treatment of envenoming by the Sri Lankan Russell's viper (Daboia Russelii Russelii): a preliminary dose-finding and pharmacokinetic study.

Russell's viper is the most important cause of life-threatening snake bite and acute renal failure in Sri Lanka. Only equine polyspecific antivenoms imported from India are available. They have not proved effective clinically or in clearing venom antigenemia and they frequently cause reactions. In an attempt to reduce mortality and morbidity, a new monospecific ovine Fab fragment antivenom (PolongaTab; Therapeutic Antibodies, Inc., London, United Kingdom) was raised against Sri Lankan Russell's viper venom. In a preliminary dose-finding study in 35 patients, an initial dose of 3-4 g restored blood coagulability permanently and stopped systemic bleeding, even in severely envenomed patients. Venom antigenemia disappeared within 1 hr of antivenom treatment but recurred, probably as a result of continued absorption of venom from the site of the bite, after the rapid clearance of therapeutic antibody. Twelve patients (34%) experienced early reactions that were usually mild and always responded to epinephrine.

Absorption and elimination of viper venom after antivenom administration.

The mechanisms by which antivenom neutralizes the venom are still poorly understood. In the present work, we studied the effects of antivenom, constituted with either F(ab')2 or Fab, on the processes of absorption and elimination of Vipera aspis venom in experimentally envenomed rabbits. We first concluded from this study that during the few hours after intramuscular injection, the venom rapidly disappeared from the site of injection but did not immediately reach the vascular system, suggesting that it is partly absorbed via the lymphatic circulation. Concerning the elimination process of the venom in the presence of antivenom, we observed that the elimination of F(ab')2/venom complexes is slower than that of free venom in the absence of antivenom but faster than that of free F(ab')2, suggesting that F(ab')2/venom complexes are eliminated by phagocytosis. The Fab/venom complexes, on the other hand, are eliminated more slowly than free Fab. These complexes are not eliminated through the renal route in agreement with their high molecular weight. In addition, we observed that the treatment of envenomed rabbits with antivenom made of Fab, but not F(ab')2, is responsible for an oliguria that could be responsible for clinical problems.

Preclinical assessment of immunoreactivity of a new purified equine F(ab')2 against European viper venom.

The immunological and pharmacokinetic properties of a new, further purified, pasteurized preparation of equine F(ab')2 (VIPERFAV) against Vipera aspis, Vipera berus, and Vipera ammodytes venom were compared with the current equine F(ab')2 preparation (IPSER Europe). Affinity constants of the V. aspis-specific F(ab')2 were determined using biosensor technology and found to be in the range of 10(8) M-1 for the four antigenic fractions of V. aspis toxins and for both F(ab')2 preparations. The improvement of 51% in the specific activity (LD50 mg-1) of the new F(ab')2 was in close agreement with the 1.8-fold increase in the immunoreactive fraction of the new preparation. In vivo investigations of venom immunocomplexation by F(ab')2 in rabbits confirmed the ability of F(ab')2 to neutralize and redistribute toxin venom. Infusion of a stoichiometric molar ratio (i.e., 1 mg kg-1) of the new antivenom induced a 2.3-fold elevation of the plasma venom concentration with a Tmax observed 8 h after F(ab')2 administration and a decline in the terminal half-life from 31.92 +/- 4.49 h to 16.73 +/- 4.34 h, in contrast, for the venom alone. The area under the curve was 1.4-fold greater in the VIPERFAV group than in the IPSER Europe group during the post-F(ab')2 infusion period. Increasing the F(ab')2 dose to 3 mg kg-1 increased by 27% the percent of venom bound to F(ab')2. Finally, the greater the venom distribution, the smaller and less pronounced the plasma redistribution. These results demonstrate that the purification and pasteurization steps involved in the preparation of the new F(ab')2 have no deleterious influence on F(ab')2 affinity but, on the contrary, improve the protective efficacy. Alteration of viper venom kinetics by specific F(ab')2 antivenom was also shown to be dependent on the interval between of F(ab')2 administration and venom bite and on the specific F(ab')2 dose administered.

The disintegrin eristostatin interferes with integrin alpha 4 beta 1 function and with experimental metastasis of human melanoma cells.

Peptides containing the integrin recognition sequence, RGD, can inhibit experimental metastasis of mouse melanoma cells, but the integrin(s) affected in these experiments is unknown. Besides "classical" RGD-binding integrins such as alpha 5 beta 1 and alpha v beta 3, RGD has been reported to bind alpha 4 beta 1, and mAbs to alpha 4 beta 1 can inhibit melanoma metastasis. We investigated the mode of action of the disintegrin eristostatin, an RGD-containing peptide isolated from snake venom, in a human melanoma experimental metastasis model. Lung colonization following i.v. injection of MV3 cells in nude mice was strongly inhibited by eristostatin. MV3 cells bound FITC-eristostatin and adhered to eristostatin-coated wells. This adhesion was partially inhibited by a GRGDSP peptide and by alpha 4 mAb. Binding of FITC-eristostatin to Jurkat cells and adhesion of Jurkat (but not K562) cells to eristostatin-coated wells further suggested that eristostatin binds alpha 4 beta 1, even though, again, alpha 4 mAb only partially inhibited adhesion. Expression of alpha 4 beta 1 was enhanced in metastatic melanoma cells compared to normal melanocytes and nonmetastatic melanoma cells. Finally, eristostatin inhibited adhesion of both MV3 and CHO alpha 4 cells to the alpha 4 beta 1-ligand VCAM-1, while adhesion to other ligands via other integrins was not affected. These findings demonstrate that inhibition of melanoma cell metastasis by RGD-containing peptides such as eristostatin, may be due to interference with alpha 4 beta 1-VCAM binding, in addition to inhibition of the classical RGD-binding integrins.

Kinetic study of Russell's viper venom in envenomed patients.

Serum levels of Russell's viper venom in 30 patients bitten by Russell's viper were measured by an ELISA. In the initial serum samples, which were collected immediately after admission to the hospital (0.5-19 hr after the bite), venom was detected in 24 patients (80%), with levels ranging from 3 to 92 ng/ml. These levels correlated well with the patient's clinical signs. At 6-12 hr after antivenom therapy, the venom levels in most of the serum samples (21 of 24, 87%) had decreased to undetectable levels. In the remaining patients, whose serum venom levels could be detected 36-72 hr after therapy, the levels varied according to the effect of the antivenom therapy and the release of venom from a deposit at the bite site.

Effect of antivenom on venom pharmacokinetics in experimentally envenomed rabbits: toward an optimization of antivenom therapy.

Antivenomous immunotherapy is still used empirically. To improve the efficacy and safety of immunotherapy, we studied the effects of administering antivenom antibodies (F(ab')2) on the pharmacokinetics of the Vipera aspis venom in rabbits. Free venom levels were measured by enzyme-linked immunosorbent assay and total concentrations were quantified by measuring the radioactivity of trichloroacetic acid-precipitable radioiodinated venom. The intravenous infusion of 125 mg of antivenom 7 h after intramuscular injection with 700 microg x kg(-1) of V. aspis venom produced a redistribution of the venom antigens from the extravascular to the vascular space. Moreover, anti-venom antibodies were able to neutralize the totality of venom antigens in the vascular space, because no free plasma venom was detectable by enzyme-linked immunosorbent assay within 15 min after antivenom injection. Similar effects were obtained after injection of 25 mg of antivenom; however, the venom was only partially neutralized with lower doses (5 and 2.5 mg). We further established that intravenous injection is the most efficient route for antivenom administration, and we examined the effects of early and late immunotherapy. Finally, the efficacy of Fab antibodies was compared with that of F(ab')2; the plasma redistribution and the immunoneutralization of the venom were lower than those induced after injection of the same dose of F(ab')2. The difference between the effects of F(ab')2 and Fab could be explained by the differential pharmacokinetics of the two fragments.

The efficacy of compression immobilization technique in retarding spread of radio-labeled Russell's viper venom in rhesus monkeys and 'mock venom' NaI131 in human volunteers.

The efficacy of the modified compression immobilization technique in retarding spread of radio-labeled Russell's viper venom in 3 rhesus monkeys (Macaca mulata) and "mock venom" NaI131 in 14 human volunteers was studied. 0.1 microgram of Russell's viper venom having 10 microCi radioactivity in 0.2 ml normal saline containing 0.5% bovine serum albumin was injected subcutaneously at the lateral aspect of the right hind limb of a rhesus monkey. A hand-tight bandaging of a rubber pad measuring 55 x 28 x 16 mm over the injection site and splinting effectively retard spread of radio-labeled venom for the entire length of time applied, although complete immobilization was not achieved. In human volunteers, application of a pad measuring 60 x 50 x 17 mm over the subcutaneous injection site of 20 microCi or 12 microCi/0.2 ml NaI131 with a hand-tight bandaging (60 +/- 10 mmHg) and immobilization of limb was found to be effective in retarding the movement of radioactive NaI131. These results suggested that the compression pads tried in this study effectively retard the spread of radio-labeled Russell's viper venom (MW ranging from 20,000-90,000) and radioactive NaI131 (MW 150) from the site of injection. Thus, it is highly likely that the present compression pad will be useful as a first-aid measure in Russell's viper bite victims.

Pharmacokinetics of Vipera aspis venom after experimental envenomation in rabbits.

Toxicokinetic studies of Vipera aspis venom were performed in rabbits after experimental envenomation. Venom proteins with a molecular weight greater than 6 kDa (high-molecular weight proteins) and which reacted in enzyme-linked immunosorbent assay with specific antiviper venom Fab'2, were also responsible for the lethal potency and the capillary permeability increasing activity of the venom. Conversely, low-molecular weight proteins were not detected by enzyme-linked immunosorbent assay and were pharmacologically inactive. The toxicokinetics of both classes of venom components were studied, using high-molecular weight and low-molecular weight radiolabeled proteins as well as enzyme-linked immunosorbent assay. After intravenous injection, Vipera aspis venom in plasma followed a biexponential decline with a distribution half-life of 0.7 hr and an elimination half-life of 12 hr. The distribution volume was 1.2 l.kg-1 and the systemic clearance was 84 ml.hr-1.kg-1. Venom levels in plasma after intramuscular injection of three doses (300, 500 and 700 micrograms/kg) of venom increased within the few hours after the venom administration to reach maximal values proportional to the injected doses. They subsequently followed a monoexponential decline, with an apparent terminal half-life of 32.5 hr. Absorption was a kinetically complex process, rapid during the first 24 hr and continued at a slower rate over the subsequent 72 hr. Bioavailability of venom was about 65%, regardless of the administered dose, and less than 5% of venom injected was excreted by the renal route.

Purification and partial characterization of a haemorrhagin (VRH-1) from Vipera russelli russelli venom.

A haemorrhagic toxin (VRH-1) has been purified to homogeneity from Vipera russelli russelli venom by subjecting it to chromatography twice successively on CM-Sephadex C-50. It is a protein of mol. wt 22,000 and contains one mole of Mg2+. Intradermal administration of this haemorrhagin in mice resulted in severe lung haemorrhage but produced little haemorrhage in skin. This apparent organ preference led us to develop a new haemorrhage assay method utilizing dye diffusion from lung in vitro. Proteolytic activity of VRH-1 was demonstrated using dimethylcasein as substrate following quantitation by reaction with trinitrobenzoyl sulfonic acid. Both haemorrhagic and proteolytic activities of VRH-1 were inhibited by serine protease inhibitors like phenylmethyl sulfonyl fluoride and chymostatin, but metal chelators had no effect. Lung haemorrhage is unlikely to be a direct reflection of a high local concentration of VRH-1. The administration of supernatant generated by incubation of chopped liver from untreated mouse and VRH-1 (in subhaemorrhagic dose) results in severe lung haemorrhage. This raises the possibility that VRH-1 leads to the formation of intermediate(s) which causes the haemorrhage.

Regional extraction of endothelins and conversion of big endothelin to endothelin-1 in the pig.

Endothelin-like immunoreactivity (ET-LI), mean arterial blood pressure (MABP) and vascular resistance in the spleen, kidney and femoral vascular bed were measured during intravenous infusions (20 pmol.kg-1.min) of endothelin-2 (ET-2), endothelin-3 (ET-3), big endothelin-1 (big ET) and sarafotoxin 6b in the pig. All peptides (especially endothelin-2 and sarafotoxin 6b) caused vasoconstrictor effects in the kidney. Endothelin-2, endothelin-3 and sarafotoxin 6b also evoked significant increases in splenic and femoral vascular resistance. The relative vasoconstrictor response to endothelin-2 was larger in the kidney and spleen than in the femoral vascular bed whereas the opposite was observed for endothelin-3. A high degree of plasma clearance for endothelin-like immunoreactivity was observed. Thus, for ET-2 and ET-3 about 70% of arterial endothelin-like immunoreactivity was removed over the kidney while over the spleen and femoral vascular bed an extraction of 50% for plasma endothelin-2 and 30-40% for endothelin-3 was observed. Big endothelin-1 was only extracted by 34% over the kidney and not at all in the splenic or femoral vascular bed. The metabolic plasma half-lifes of endothelin-2 and endothelin-3 in vivo were in the same range, 1-2 minutes, whereas the half-life of big endothelin-1 was 9 minutes. HPLC-characterization of the plasma endothelin-like immunoreactivity during and after big endothelin-1 infusion as well as measurements using a specific endothelin-1 antiserum revealed formation of endothelin-1 from circulating big endothelin-1 in vivo but not in plasma in vitro. It is concluded that there exists specificity concerning the vasoconstrictor effects and the removal of endothelin-peptides from the circulation, both mechanisms being most prominent in the kidney. Big endothelin-1 has a much longer metabolic half-life, less regional clearance and poor vasoconstrictor activity compared with endothelin-1. Furthermore, endothelin-1 is formed from circulating big endothelin-1 probably by an endothelin-converting enzyme.