Effects of antivenom on Buthus occitanus tunetanus (Bot) scorpion venom pharmacokinetics: towards an optimization of antivenom immunotherapy in a rabbit model.

The pharmacokinetic parameters of Bot venom were determined in a rabbit model using a specific sandwich type ELISA. After intravenous injection, Bot venom seems to follow a three-compartment pharmacokinetic open model. However, after subcutaneous injection, the distribution and elimination kinetics of Bot venom are best characterized by a bi-compartment pharmacokinetic open model. Bot venom is completely absorbed from its SC injection site, since the absolute bioavailability is higher than 95%; the maximum plasma venom concentration is reached between 30 and 60 min after venom injection. Bot venom diffuses rapidly to tissues and is distributed in a high body volume. The total body clearance of Bot venom is relatively high in agreement with a low mean residence time. Antivenom immunotherapy experiments were carried out in the rabbit model, in order to select the most appropriate strategy for the adequate use of this treatment. The effects of the route, the dose and the delay of antivenom injection on Bot venom pharmacokinetic parameters and on the antivenom immunotherapy efficacy were then studied. These studies indicated in particular that: (1) the injection of a minimal neutralizing antivenom dose is required for a complete and permanent neutralization of circulating venom antigens; this dose is named minimal (threshold) efficacious antivenom dose; (2) the intramuscular route is not the most appropriate way for antivenom injection; and (3) a delayed antivenom immunotherapy remains efficacious especially on the neutralization of the remaining circulating venom. In short, these experimental studies show that early intravenous injection of an appropriate antivenom dose (at least the threshold efficacious dose) is the indicated way for a rapid and permanent neutralization of circulating scorpion venom toxins.

Evaluation of antivenom therapy in children severely envenomed by Androctonus australis garzonii (Aag) and Buthus occitanus tunetanus (Bot) scorpions.

One hundred and forty-seven cases of envenomed children under 15 years old presenting local and general symptoms without failure in vital functions (clinical grade II) or presenting serious general symptoms with failure in vital functions (clinical grade III) were collected during the summer seasons of 1993-1997. They were classified in six groups according to the use or not of antivenom, the route and the frequency of antivenom administration. The determination, by a sensitive ELISA, of blood venom concentration before and until 6 h after antivenom therapy, allowed the establishment of the venom toxicokinetic curve for each group. The intramuscular administration of antivenom did not show significant effects on venom toxicokinetic curves and on patients recovery time. However, the same amount of antivenom administered by intravenous route clear rapidly the blood free venom toxins. Also, the patient recovery time was significantly shortened. These data are in favor of intravenous application of an adequate dose of an efficient antivenom in order to treat successfully severe scorpion envenoming cases.

Anti-platelet activity of the peptides composing the lebetin 1 family, a new class of inhibitors of platelet aggregation.

We have purified from Vipera lebetina venom a family of inhibitors of platelet aggregation, named Lebetins. They are composed of two peptide groups of short (Lebetin 1: L1alpha: GDNKPPKKGPPNG; L1beta: DNKPPKKGPPNG) and long (Lebetin 2: L2alpha: GDNKPPKKGPPNGCFGHKIDRIGSHSGLGCNKVDDNKG; L2beta: DNKPPKKGPPNGCFGHKIDRIGSHSGLGCNKVDDNKG) size. The sequence presenting anti-platelet activity is mainly present within the Lebetin 1 sequence [Barbouche, R. Marrakchi, N., Mansuelle, P., Krifi, M., Fenouillet, E., Rochat, H. and El Ayeb, M. (1996) Novel anti-platelet aggregation polypeptides from Vipera lebetina venom: isolation and characterization. FEBS Lett. 392, 6-10]. Here, the peptides that compose the Lebetin 1 family were synthesized. Their respective activity was determined. Synthetic L1alpha and L1beta inhibited collagen-induced platelet aggregation in the nanomolar range. A peptide corresponding to L1beta deleted by D at its N terminus (L1gamma) also inhibited platelet aggregation potently; further truncation of L1gamma impaired its activity. Because L1 peptides efficiently inhibited fibrinogen-induced alpha-chymotrypsin treated-platelet aggregation, we tested whether they act mainly through the inhibition of platelet binding to fibrinogen and showed that they failed to inhibit platelet binding to fibrinogen-coated wells. The activity of L1 peptides was also tested in vivo: their intravenous administration strongly inhibited collagen-induced thrombocytopenia in rats.

Development of an ELISA for the detection of scorpion venoms in sera of humans envenomed by Androctonus australis garzonii (Aag) and Buthus occitanus tunetanus (Bot): correlation with clinical severity of envenoming in Tunisia.

A sandwich ELISA was set up for measuring scorpion venom levels in sera of accidentally envenomed humans with the aim to establish a quantitative relationship between these levels, envenoming severity and clinical symptoms. This assay used equine polyclonal F(ab')2, specific to two North African scorpion (Androctonus australis garzonii: Aag and Buthus occitanus tunetanus: Bot) venoms. The test proved to be simple, reproducible, very sensitive (detection limit = 0.9 ng/ml) and linear between 0.5 and 15 ng/ml of venom concentrations. A large survey on scorpion sting envenomings was conducted from 1993 to 1996 in Tunisia to gather accurate epidemiological, clinical and biological data from victims as well as informations on the treatment that they had received. Victims were classified into three grades (GI, GII and GIII) of increasing severity according to clinical signs of envenoming. Blood samples were collected from victims and tested by ELISA for their content of Aag and Bot venoms. A strong correlation was found between clinical symptoms of envenoming and the level of scorpion venom antigens in serum (r = 0.980). Mean serum venom concentrations were: 2.65 +/- 0.81 ng/ml in GI envenoming, 9.79 +/- 4.08 ng/ml in GII and 21.7 +/- 6.51 ng/ml in GIII. The difference between each group was statistically significant (p < 0.01). This ELISA may prove to be helpful to establish a rationale approach of specific antivenom therapy.

Effect of some variables on the in vivo determination of scorpion and viper venom toxicities.

An adequate assessment of scorpion and snake venom LD50 is an important step for accurate evaluation of antivenom sera potencies and the optimization of serotherapy. The LD50 variation of Tunisian scorpion (Androctonus australis garzonii: Aag and Buthus occitanus tunetanus: Bot) venoms with body weight, sex and strain (Swiss or C57BI/6) of mice used, the route of venom injection, the venom-milking procedures (manually or electrically) and the venom batches have been studied over a 7-year period (1990-1996). Aag venom is 3-4 times more toxic than Bot venom. However for both venoms, the LD50 determined in C57BI/6 mice, in small body weight animal or by intraperitoneal route were respectively significantly lower than those determined in Swiss mice, in high body weight or by subcutaneous route. Significant LD50 variations (25-50%) were also seen from one electrically prepared batch to another. A good correlation (r = 0.982) was observed between the concentrations of the crude venom toxic fraction determined by ELISA and LD50 values when assessed in vivo. The LD50 variation of Tunisian viper (Cerastes cerastes: Cc and Vipera lebetina: VI) venoms with the strain (Swiss or BALB/c), sex and body weight of mice used, the season and the year of venom milking were also investigated over a 3-year period (1990-1992). No significant LD50 variations were observed with the mouse strain, the sex or the season of venom milking. However, LD50 varies significantly with the year of the venom collection and the body weight of mice used. Furthermore, SDS-PAGE analysis shows annual variation for VI venom composition where no such variations were observed for Cc venom. These results stress the need either for the standardization of the venom LD50 evaluation or of the venom quality used for the development of an efficient antivenom.

An equilibrium ELISA for the dosage of Androctonus australis garzonii (Aag) and Buthus occitanus tunetanus (Bot) scorpion venoms: set up and calibration.

Scorpion stings are very frequent in Tunisia; yet a method for evaluating envenoming severity and consequently victim treatment, has never been adequately established nor has its efficiency been properly evaluated. Indeed, a management of envenomed patients requires the optimization of envenoming antivenom immunotherapy. This task requires either, an accurate evaluation of toxicokinetic parameters of scorpion venoms in absence and in presence of antivenom, using animals as models, and the establishment of a quantitative relationship between human blood scorption venom levels, envenoming severities and clinical symptoms. A performant sandwich ELISA was set up and calibrated for measuring scorpion venom levels in human and rabbit sera. This assays was performed with polyclonal F(ab')2 specific to the two North African scorpion (Androctonus australis garzonii; Aag and Buthus occitanus tunetanus: Bot) venoms. It is simple, rapid, very sensitive (detection limit = 0.9 ng/ml) and shows a good linearity for venom concentrations in human sera comprised between 0.5 and 15 ng/ml. The ELISA is also reproducible: the coefficient of variation, determined at different venom concentrations (low: 4 ng/ml; medium: 8 ng/ml and high: 12 ng/ml) prepared in a pool of sera collected from several healthy donors, were lower than 10%. Such an ELISA has been successfully used, either in experimental toxinokinetic and immunotherapeutic studies carried out in rabbits or for the quantification of Aag and Bot venom levels in the serum of human victims stung by these scorpion.

New procedures and parameters for better evaluation of Androctonus australis garzonii (Aag) and Buthus occitanus tunetanus (Bot) scorpion envenomations and specific serotherapy treatment.

New procedures describing intoxication with variable amounts of scorpion venoms (from 1 to 5 LD50) allowed us to introduce new parameters to evaluate Aag and Bot envenomations. Significant differences between the fatal limit time (FLT) and the last mortality time (LMT) were observed when the amount of Aag and Bot venom injected was equal to 1 LD50 and equal to or higher than 2 LD50. For Aag and Bot, the percentage of the fast mortality (FM) and the delayed mortality (DM) varied conversely when the amount of injected venom increased from 1 to 5 LD50. The relationship between the venom LD50 (from 2 to 20), the median protective dose (PD50) and the neutralizing activity of specific antivenom have been established. PD50 increased in a parallel manner with LD50. The neutralizing titres (LD50/ml) of Aag antivenom decreased from 74 +/- 3 to 44 +/- 2 and that of Bot antivenom from 52 +/- 2 to 36 +/- 1 when the number of LD50 injected increased from 2 to 20. Antivenom potency was evaluated using different protocols based on the presence or the absence of preincubation of the venom with the antivenom. In experiments where venom and antivenom were simultaneously but immediately injected, PD50 were twice as high as those found when venom and antivenom were preincubated (30 min at 37 degrees C). On the contrary, the corresponding neutralizing titres were two times lower. In an attempt to simulate accidental envenomations and subsequent serotherapy, Aag and Bot venom (4 LD50) were subcutaneously injected and the appropriate PD50S of antivenom were intravenously administered at different time intervals after envenomation. When the time of antivenom administration was shorter than the FLT, all envenomed mice might be protected by increasing volume of antivenom. However, when the antivenom is injected closer to the FLT only 50 to 60% of mice envenomed, respectively, by Aag and Bot could be saved even when more than 5 PD50 were injected.

Release of aldosterone and corticosterone from the adrenal cortex of the Brattleboro rat in response to administration of ACTH.

The time-course and dose-response of the in-vivo secretion of aldosterone and corticosterone after administration of ACTH(1-24) were measured in adrenal venous blood from female Brattleboro rats, homozygous for hypothalamic diabetes insipidus and lacking arginine vasopressin (AVP). Female Long-Evans rats were used as controls. All animals were pretreated with dexamethasone and anaesthetized with pentobarbital. Basal secretions of aldosterone and corticosterone were four- to sixfold lower in Brattleboro than in Long-Evans rats. Administration of ACTH consistently increased the secretion of aldosterone and corticosterone similarly in the two groups of rats; maximum values were observed 20-30 min after ACTH injection. However, for all the doses of ACTH (0.05, 0.5 and 5.0 mi.u./100 g body wt) and at every stage of response the secretion rates of aldosterone and corticosterone were twofold lower in Brattleboro than in Long-Evans rats. Furthermore the absolute increase in steroid secretion induced by ACTH was reduced by half in Brattleboro rats. These results show that the impairment of adrenal activity is largely due to a reduced capacity for corticosteroidogenesis in the adrenal cortex of Brattleboro rats. The mechanisms of action of AVP are discussed.

[Secretion of aldosterone and corticosterone by the adrenals of Brattleboro rats in response to ACTH administration]. / Sécrétions d'aldostérone et de corticostérone par la surrénale du Rat Brattleboro en réponse à l'administration d'ACTH.

The secretions of aldosterone and corticosterone in response to administration of 0,5 mUI of (1,24) ACTH (synacthène-Ciba) were measured in the adrenal venous blood of 15 Brattleboro female rats genetically lacking vasopressin and in 15 Long-Evans female rats, pretreated with dexamethasone. The secretions of aldosterone and corticosterone increased according to a similar profile in the two groups of animals: maximum values were 20-30 min. after ACTH injection; however the steroidogenic secretion of the adrenal cortex was always about 50% less in the Brattleboro female rats than in Long-Evans female rats. This result suggests mainly that vasopressin may be involved in the mechanisms which control the in vivo production of aldosterone by the adrenal glomerulosa cells.